CN117120072A - Methods of treating aging-related disorders - Google Patents

Abstract

Provided herein are methods of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells and diseased cells in a subject in need thereof, reducing the accumulation of naturally occurring and/or treatment-induced senescent cells and diseased cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators and/or one or more agents that result in reduced activation of TGF-beta receptors.

Description

Methods of treating aging-related disorders

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application Ser. No. 63/032,933, filed on 1-6-2020, international patent application Ser. No. PCT/US2020/035598, filed on 1-6-2020, and U.S. provisional patent application Ser. No. 63/118,536, filed on 25-11-2020, the entire contents of which are incorporated herein by reference.

Technical Field

The present disclosure relates to the fields of immunology and cell biology.

Background

Senescence is a form of irreversible growth arrest, accompanied by phenotypic changes, resistance to apoptosis, and activation of injury-sensing signaling pathways. Cell senescence was initially described in cultured human fibroblasts, which lost their proliferative capacity, reaching permanent arrest after about 50 population doublings (known as the Hayflick limit). Senescence is thought to be a stress response that can be induced by a wide range of intrinsic and extrinsic injuries, including oxidative and genotoxic stress, DNA damage, telomere loss, oncogene activation, mitochondrial dysfunction, or chemotherapeutic agents.

Senescent cells remain metabolically active and can affect tissue hemostasis, disease, and aging through their secretory phenotype. Aging is considered a physiological process and is important to promote wound healing, tissue homeostasis, regeneration and regulation of fibrosis. For example, transient induction of senescent cells is observed during wound healing, and it contributes to wound healing. Aging also plays a role in inhibiting tumors. However, aging cell accumulation also drives aging and aging-related diseases and disorders. The senescent phenotype may also trigger a chronic inflammatory response and thus enhance a chronic inflammatory condition to promote tumor growth. The link between aging and aging was initially based on the observation that aging cells accumulated in aging tissues. The use of transgenic models has enabled systematic detection of senescent cells in many aging-related disorders. Strategies to selectively eliminate senescent cells suggest that senescent cells play a causal role in aging-related disorders.

Cell senescence is a series of progressive and phenotypically diverse cell states obtained after initial growth arrest (van Deursen, nature 509 (7501): 439-446, 2014), and thus senescent cells are heterogeneous cell populations with few shared core properties (Dou et al, nature 550 (7676): 402-406, 2017). Thus, it is difficult to determine a co-senescent cell lysis drug (senolytic drug) target. This also hampers the achievement of the goal of developing senolytics, a method of selectively, safely and effectively eliminating senescent cells after systemic administration. As described above, immune cells are effector cells that naturally eliminate senescent cells after they have completed their physiological actions. (Brighton et al, elife 6, 2017). Attenuation of the immune system during aging results in the accumulation of aging cells (Karin et al, nat. Comm.10 (1): 5495, 2019; chambers et al, J. Allergy Clin. Immunol.145 (5): 1323-1331, 2020).

Disclosure of Invention

The invention is based on the following findings: subcutaneous administration of an agent that results in reduced activation of TGF- β receptors or a common gamma chain family cytokine receptor activator (e.g., a complex of gamma chain cytokines and their cognate receptors) to a mammal will promote and activate immune cells to regain their ability to effectively, selectively and safely reduce senescent cells in vivo. In view of this discovery, provided herein are methods of killing or reducing the number of naturally occurring and/or therapeutically induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors. Also provided herein are methods of reducing naturally occurring and/or treatment-induced accumulation of senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors. Also provided herein are methods of reducing the levels of markers of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors. Also provided herein are methods of attenuating the activity of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more agents that result in attenuation of activation of a TGF- β receptor. Also provided herein are methods of reducing the level and/or activity of one or more naturally occurring and/or therapeutically induced senescence-associated secretory phenotype ("SASP") factors derived from senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

Also provided herein are methods of killing and reducing the number of naturally occurring and/or therapeutically induced senescent cells in a subject (as well as methods of reducing the accumulation of senescent cells or reducing markers of senescent cells) comprising administering to a subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators (e.g., complexes of gamma chain cytokines and their cognate receptors). Also provided herein are methods of reducing the activity of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. Also provided herein are methods of reducing the level and/or activity of one or more SASP factors derived from naturally occurring and/or therapeutically induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

The invention is also based on the following findings: administration of NK cell activators to mammals with cancer results in tumor inhibition, and administration of NK cell activators to diabetic animal models shows improved skin and hair appearance and texture, as well as reduced blood glucose levels. In view of this discovery, provided herein are methods of treating an aging-related disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of one or more Natural Killer (NK) cell activators and/or a therapeutically effective amount of activated NK cells to a subject identified as having an aging-related disease or disorder. Also provided herein are methods of killing or reducing the number of senescent cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more NK cell activators and/or a therapeutically effective amount of activated NK cells. Also provided herein are methods of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators and/or a therapeutically effective amount of activated NK cells. Also provided herein are methods of aiding in the treatment of obesity in a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators and/or a therapeutically effective amount of activated NK cells.

Provided herein are methods of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

Also provided herein are methods of reducing naturally occurring and/or treatment-induced accumulation of senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

Also provided herein are methods of reducing the levels of markers of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

Also provided herein are methods of attenuating the activity of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more agents that result in attenuation of activation of a TGF- β receptor.

Also provided herein are methods of reducing the level and/or activity of one or more SASP factors derived from naturally occurring and/or therapeutically induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of a TGF-beta receptor.

In some embodiments of any of the methods described herein, the subject has been diagnosed or identified as having an aging-related disease or inflammatory disease. In some embodiments of any one of the methods described herein, the aging-related disorder is inflammatory aging-related. In some embodiments of any one of the methods described herein, the aging-related disorder is selected from the group consisting of: alzheimer's disease, aneurysms, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, inflammatory bowel disease, intervertebral disc degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, parkinson's disease, age-related loss of elasticity of lung tissue, age-related macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction. In some embodiments of any one of the methods described herein, the aging-related disorder is a cancer selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments of any one of the methods described herein, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, crohn's disease, multiple sclerosis, guillain barre syndrome, psoriasis, grave's disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders and cognitive disorders associated with cancer treatment.

In some embodiments of any one of the methods described herein, the treatment-induced senescent cell is a chemotherapy-induced senescent cell. In some embodiments of any of the methods described herein, administration of one or more agents that result in reduced TGF- β receptor activation results in a reduction in the number or activity of naturally occurring senescent cells and/or treatment-induced senescent cells in the target tissue of the subject. In some embodiments of any one of the methods described herein, the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vasculature, skeletal tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

In some embodiments of any of the methods described herein, the tgfβ receptor is TGF- β receptor II (tgfβrii). In some embodiments of any one of the methods described herein, the tgfβ receptor is tgfβriii.

In some embodiments of any of the methods described herein, at least one of the one or more agents that result in reduced activation of the TGF- β receptor is a soluble TGF- β receptor, an extracellular domain of the TGF- β receptor, an antibody that specifically binds TGF- β, an antagonistic antibody that binds to the TGF- β receptor, an agent that binds to latency-related peptide ("LAP"), or an agent that binds to a TGF- β/LAP complex. In some embodiments of any of the methods described herein, the one or more agents that result in reduced TGF- β receptor activation reduce TGF- β receptor activation by binding to LAP or TGF- β/LAP complex.

In some embodiments of any of the methods described herein, at least one of the one or more agents that result in reduced TGF- β receptor activation is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain; (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; (ii) A second target binding domain, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a ligand of a TGF- β receptor; or one or both of the first target binding domain and the second target binding domain is an antagonistic antigen binding domain that specifically binds to a TGF- β receptor. In some embodiments of any one of the methods described herein, the TGF- β receptor is tgfbetarii. In some embodiments of any one of the methods described herein, the TGF- β receptor is tgfbetariii.

In some embodiments of any of the methods described herein, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of any of the methods described herein, the soluble tissue factor domain and the first domain in the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

In some embodiments of any of the methods described herein, the second domain in a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises one or more additional target binding domains.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93.

In some embodiments of any of the methods described herein, the pair of affinity domains are a sushi domain from the alpha chain of the human IL-15 receptor (il15rα) and soluble IL-15. In some embodiments of any of the methods described herein, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments of any of the methods described herein, the soluble IL-15 comprises a mutation that reduces or eliminates IL-15 activity.

In some embodiments of any one of the methods described herein, the pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, and SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25. In some embodiments of any of the methods described herein, the first domain or the second domain of the pair of affinity domains is a soluble common gamma chain family cytokine or an antigen binding domain that specifically binds to a common gamma chain family cytokine receptor.

In some embodiments of any of the methods described herein, the first target binding domain and/or the second target binding domain comprises a soluble TGF- β receptor. In some embodiments of any of the methods described herein, the soluble TGF- β receptor is soluble tgfbetarii. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a first sequence that is at least 80% identical to SEQ ID NO. 183, and a second sequence that is at least 80% identical to SEQ ID NO. 183, wherein the first and second sequences are separated by a linker. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a first sequence that is at least 90% identical to SEQ ID NO. 183, and a second sequence that is at least 90% identical to SEQ ID NO. 183. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a first sequence of SEQ ID NO. 183 and a second sequence of SEQ ID NO. 183. In some embodiments of any of the methods described herein, the linker comprises the sequence of SEQ ID NO. 102. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a sequence at least 80% identical to SEQ ID NO 188. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a sequence at least 90% identical to SEQ ID NO 188. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises the sequence of SEQ ID NO: 188.

In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 236. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID No. 236. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO. 236. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 193. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 236. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO 193. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises the sequence of SEQ ID NO: 193. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO. 236.

In some embodiments of any of the methods described herein, at least one of the one or more agents that result in reduced TGF- β receptor activation is a single chain chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a second target binding domain, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a ligand of a TGF- β receptor; or one or both of the first target binding domain and the second target binding domain is an antagonistic antigen binding domain that specifically binds to a TGF- β receptor. In some embodiments of any one of the methods described herein, the TGF- β receptor is tgfbetarii. In some embodiments of any one of the methods described herein, the TGF- β receptor is tgfbetariii.

In some embodiments of any of the methods described herein, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain. In some embodiments of any of the methods described herein, the soluble tissue factor domain and the second target binding domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises one or more additional target binding domains located at its N-terminus and/or C-terminus. In some embodiments of any of the methods described herein, the first target binding domain and/or the second target binding domain comprises a soluble TGF- β receptor. In some embodiments of any of the methods described herein, the soluble TGF- β receptor is soluble TGF- βrii.

In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a first sequence that is at least 80% identical to SEQ ID NO. 183, and a second sequence that is at least 80% identical to SEQ ID NO. 183, wherein the first and second sequences are separated by a linker. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a first sequence that is at least 90% identical to SEQ ID NO. 183, and a second sequence that is at least 90% identical to SEQ ID NO. 183. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a first sequence of SEQ ID NO. 183 and a second sequence of SEQ ID NO. 183. In some embodiments of any of the methods described herein, the linker comprises the sequence of SEQ ID NO. 102. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a sequence at least 80% identical to SEQ ID NO 188. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises a sequence at least 90% identical to SEQ ID NO 188. In some embodiments of any of the methods described herein, the soluble TGF-beta RII comprises the sequence of SEQ ID NO: 188.

In some embodiments of any of the methods described herein, the method comprises administering to the subject two or more doses of one or more agents that result in reduced activation of the TGF- β receptor. In some embodiments of any of the methods described herein, any two or more doses are administered at intervals of about 1 week to about one year. In some embodiments of any of the methods described herein, any two consecutive doses of the two or more doses are administered between about 1 week and about 6 months apart. In some embodiments of any of the methods described herein, any two consecutive doses of the two or more doses are administered between about 1 week and about 2 months apart. In some embodiments of any of the methods described herein, any two consecutive doses of the two or more doses are administered between about 1 week and about 1 month apart.

In some embodiments of any of the methods described herein, the two or more doses are administered by subcutaneous administration. In some embodiments of any of the methods described herein, the two or more doses are administered by intramuscular injection.

In some embodiments of any of the methods described herein, two or more doses are administered over a period of about 1 year to about 60 years. In some embodiments of any of the methods described herein, two or more doses are administered over a period of about 1 year to about 50 years. In some embodiments of any of the methods described herein, two or more doses are administered over a period of about 1 year to about 40 years. In some embodiments of any of the methods described herein, two or more doses are administered over a period of about 1 year to about 30 years. In some embodiments of any of the methods described herein, two or more doses are administered over a period of about 1 year to about 20 years. In some embodiments of any of the methods described herein, two or more doses are administered over a period of about 1 year to about 10 years.

In some embodiments of any of the methods described herein, the one or more agents that result in reduced TGF- β receptor activation of the first agent begin when the subject reaches at least 30 years of age. In some embodiments of any of the methods described herein, the one or more agents that result in reduced TGF- β receptor activation of the first agent begin when the subject reaches at least 40 years of age. In some embodiments of any of the methods described herein, the one or more agents of the first dose that result in reduced TGF- β receptor activation begin when the subject reaches at least 50 years of age. In some embodiments of any of the methods described herein, the one or more agents of the first dose that result in reduced TGF- β receptor activation begin when the subject reaches at least 60 years of age.

In some embodiments of any of the methods described herein, each of the two or more doses is administered at a dose of about 0.01mg/kg of each agent that results in reduced TGF- β receptor activation to about 10mg/kg of each agent that results in reduced TGF- β receptor activation. In some embodiments of any of the methods described herein, each of the two or more doses is administered at a dose of about 0.02mg/kg of each agent that results in reduced TGF- β receptor activation to about 5mg/kg of each agent that results in reduced TGF- β receptor activation.

In some embodiments of any one of the methods described herein, the subject has not been diagnosed or identified as having an aging-related disease or inflammatory disease. In some embodiments of any one of the methods described herein, the subject has not been previously treated with a chemotherapeutic agent. In some embodiments of any of the methods described herein, the subject has not been previously treated with a therapeutic agent that induces cellular senescence.

Provided herein are methods of treating an aging-related disease or disorder in a subject in need thereof, comprising administering to a subject identified as having an aging-related disease or disorder a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Also provided herein are methods of killing or reducing the number of senescent cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more NK cell activators. In some embodiments of any of the methods described herein, the senescent cell is a senescent cancer cell, a senescent monocyte, a senescent lymphocyte, a senescent astrocyte, a senescent microglial cell, a senescent neuron, a senescent tissue fibroblast, a senescent dermal fibroblast, a senescent keratinocyte, or other differentiated tissue-specific dividing functional cell. In some embodiments of any one of the methods described herein, the senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced senescent cells. In some embodiments of any one of the methods described herein, the subject has been identified or diagnosed as having an aging-related disease or disorder.

In some embodiments of any one of the methods described herein, the aging-related disease or disorder is selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, skin diseases, premature senility diseases and fragile diseases. In some embodiments of any one of the methods described herein, the cancer is selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of any one of the methods described herein, the autoimmune disease is type 1 diabetes.

In some embodiments of any one of the methods described herein, the metabolic disease is selected from the group consisting of: obesity, lipodystrophy and type 2 diabetes.

In some embodiments of any one of the methods described herein, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease and dementia.

In some embodiments of any one of the methods described herein, the cardiovascular disease is selected from the group consisting of: coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

In some embodiments of any one of the methods described herein, the dermatological disorder is selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo, thinning of skin, xeroderma pigmentosum and congenital dysplastic keratosis.

In some embodiments of any one of the methods described herein, the premature senility disease is selected from the group of: premature senility and Huzison-Ji Erfu premature senility syndrome.

In some embodiments of any one of the methods described herein, the fragile disease is selected from the group consisting of: friability, vaccination response, osteoporosis and sarcopenia.

In some embodiments of any one of the methods described herein, the aging-related disease or disorder is selected from the group consisting of: osteoarthritis, lipoatrophy, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related kidney dysfunction, and chemically induced kidney dysfunction.

In some embodiments of any one of the methods described herein, the aging-related disease or disorder is type 2 diabetes or atherosclerosis.

In some embodiments of any one of the methods described herein, the administering results in a reduction in the number of senescent cells in the target tissue of the subject. In some embodiments of any one of the methods described herein, the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature, skeletal tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

In some embodiments of any one of the methods described herein, administration results in increased expression levels of CD25, CD69, mTORC1, SREBP1, IFN- γ, and granzyme B in the activated NK cells.

Also provided herein are methods of treating an aging-related disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of activated NK cells to a subject identified as having an aging-related disease or disorder.

Also provided herein are methods of killing or reducing the number of senescent cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated NK cells. In some embodiments of any of the methods described herein, the senescent cell is a senescent cancer cell, a senescent monocyte, a senescent lymphocyte, a senescent astrocyte, a senescent microglial cell, a senescent neuron, a senescent tissue fibroblast, a senescent dermal fibroblast, a senescent keratinocyte, or other differentiated tissue-specific dividing functional cell. In some embodiments of any one of the methods described herein, the senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced senescent cells. In some embodiments of any one of the methods described herein, the subject has been identified or diagnosed with an aging-related disease or disorder.

In some embodiments of any one of the methods described herein, the aging-related disease or disorder is selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, skin diseases, premature senility diseases and fragile diseases. In some embodiments of any one of the methods described herein, the cancer is selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments of any one of the methods described herein, the autoimmune disease is type 1 diabetes.

In some embodiments of any one of the methods described herein, the metabolic disease is selected from the group consisting of: obesity, lipodystrophy and type 2 diabetes.

In some embodiments of any one of the methods described herein, the neurodegenerative disease is selected from the group consisting of: alzheimer's disease, parkinson's disease and dementia.

In some embodiments of any one of the methods described herein, the cardiovascular disease is selected from the group consisting of: coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

In some embodiments of any one of the methods described herein, the dermatological disorder is selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo, thinning of skin, xeroderma pigmentosum and congenital dysplastic keratosis.

In some embodiments of any one of the methods described herein, the premature senility disease is selected from the group of: premature senility and Huzison-Ji Erfu premature senility syndrome.

In some embodiments of any one of the methods described herein, the fragile disease is selected from the group consisting of: friability, vaccination response, osteoporosis and sarcopenia.

In some embodiments of any one of the methods described herein, the aging-related disease or disorder is selected from the group consisting of: age-related macular degeneration, osteoarthritis, lipoatrophy, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related kidney dysfunction, and chemically induced kidney dysfunction.

Some embodiments of any of the methods described herein further comprise: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of any one of the methods described herein, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of any one of the methods described herein, the resting NK cells are allogeneic resting NK cells. In some embodiments of any one of the methods described herein, the resting NK cells are artificial NK cells. In some embodiments of any one of the methods described herein, the resting NK cells are haplotype resting NK cells. In some embodiments of any one of the methods described herein, the resting NK cell is a genetically engineered NK cell carrying a chimeric antigen receptor or a recombinant T cell receptor. Some embodiments of any one of the methods described herein further comprise isolating the activated NK cells, followed by administering the activated NK cells to the subject. Some embodiments of any one of the methods described herein further comprise administering to the subject after introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into resting NK cells or activating NK cells.

Also provided herein are methods of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Also provided herein are methods of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of activated NK cells. Some embodiments of any of the methods described herein further comprise: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of any one of the methods described herein, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of any one of the methods described herein, the resting NK cells are allogeneic resting NK cells. In some embodiments of any one of the methods described herein, the resting NK cells are artificial NK cells. In some embodiments of any one of the methods described herein, the resting NK cells are haplotype resting NK cells. In some embodiments of any one of the methods described herein, the resting NK cell is a genetically engineered NK cell carrying a chimeric antigen receptor or a recombinant T cell receptor. Some embodiments of any one of the methods described herein further comprise isolating the activated NK cells, followed by administering the activated NK cells to the subject.

In some embodiments of any one of the methods described herein, the method provides an improvement in the texture and/or appearance of the subject's skin over a period of time. In some embodiments of any one of the methods described herein, the method reduces the rate of wrinkle formation in the skin of the subject over a period of time. In some embodiments of any one of the methods described herein, the method results in an improvement in the skin tone of the subject over a period of time. In some embodiments of any one of the methods described herein, the method reduces age spots on the skin of the subject over a period of time. In some embodiments of any one of the methods described herein, the method results in an improvement in the skin texture of the subject over a period of time. In some embodiments of any one of the methods described herein, the method results in an improvement in the texture and/or appearance of the subject's hair over a period of time. In some embodiments of any one of the methods described herein, the method results in a decrease in the rate of white hair formation in the subject over a period of time. In some embodiments of any one of the methods described herein, the method reduces the number of white hairs in the subject over a period of time. In some embodiments of any one of the methods described herein, the method causes a decrease in the rate of hair loss in the subject over time. In some embodiments of any one of the methods described herein, the method results in an improvement in the hair texture of the subject over a period of time.

In some embodiments of any one of the methods described herein, the period of time is between about one month and about 10 years. In some embodiments of any one of the methods described herein, the method causes a decrease in the number of aged dermal fibroblasts in the skin of the subject over a period of time.

Also provided herein are methods of aiding in the treatment of obesity in a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Also provided herein are methods of aiding in the treatment of obesity in a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of activated NK cells. Some embodiments of any of the methods described herein further comprise: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of any one of the methods described herein, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of any one of the methods described herein, the resting NK cells are allogeneic resting NK cells. In some embodiments of any one of the methods described herein, the resting NK cells are artificial NK cells. In some embodiments of any one of the methods described herein, the resting NK cells are haplotype resting NK cells. In some embodiments of any one of the methods described herein, the resting NK cell is a genetically engineered NK cell carrying a chimeric antigen receptor or a recombinant T cell receptor. Some embodiments of any one of the methods described herein further comprise isolating the activated NK cells, followed by administering the activated NK cells to the subject.

In some embodiments of any one of the methods described herein, the method causes a decrease in the mass of the subject over a period of time. In some embodiments of any one of the methods described herein, the method causes a decrease in the Body Mass Index (BMI) of the subject over a period of time. In some embodiments of any one of the methods described herein, the method reduces the rate at which the subject progresses from pre-diabetes to type 2 diabetes. In some embodiments of any one of the methods described herein, the method results in a decrease in fasting serum glucose levels in the subject. In some embodiments of any one of the methods described herein, the method increases insulin sensitivity in the subject. In some embodiments of any one of the methods described herein, the method results in a reduction in the severity of atherosclerosis in the subject. In some embodiments of any one of the methods described herein, the period of time is between about two weeks and about 10 years.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators causes activation of one or more of: IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor, IL-33 receptor; receptors for CD16, CD69, CD25, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR DS1, KIR2Ds2/3, KIR2DL4, KIR2DS5 and KIR3DS 1.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the IL-2 receptor is soluble IL-2 or an agonistic antibody that specifically binds to the IL-2 receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the IL-7 receptor is soluble IL-7 or an agonistic antibody that specifically binds to the IL-7 receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the IL-12 receptor is soluble IL-12 or an agonistic antibody that specifically binds to the IL-12 receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the IL-15 receptor is soluble IL-15 or an agonistic antibody that specifically binds to the IL-15 receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the IL-21 receptor is soluble IL-21 or an agonistic antibody that specifically binds to the IL-21 receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the IL-33 receptor is soluble IL-33 or an agonistic antibody that specifically binds to the IL-33 receptor.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of the CD16 receptor is an agonistic antibody that specifically binds to the CD16 receptor.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of the CD69 receptor is an agonistic antibody that specifically binds to CD 69.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of CD25 or CD59 receptor is an agonistic antibody that specifically binds to CD25 or CD 59.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of CD352 receptor is an agonistic antibody that specifically binds to CD 352.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of the NKp80 receptor is an agonistic antibody that specifically binds to NKp 80.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of DNAM-1 receptors is an agonistic antibody that specifically binds to DNAM-1.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of the 2B4 receptor is an agonistic antibody that specifically binds to 2B 4.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause activation of the NKp30 receptor is an agonistic antibody that specifically binds to NKp 30.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the NKp44 receptor is an agonistic antibody that specifically binds to NKp 44.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the NKp46 receptor is an agonistic antibody that specifically binds to NKp 46.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of the NKG2D receptor is an agonistic antibody that specifically binds to NKG 2D.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of KIR2DS1 receptor is an agonistic antibody that specifically binds to KIR2DS 1.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of KIR2DS2/3 receptors is an agonistic antibody that specifically binds to KIR2DS 2/3.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of KIR2DL4 receptors is an agonistic antibody that specifically binds to KIR2DL 4.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of KIR2DS4 receptor is an agonistic antibody that specifically binds to KIR2DS 4.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of KIR2DS5 receptor is an agonistic antibody that specifically binds to KIR2DS 5.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause activation of KIR3DS1 receptor is an agonistic antibody that specifically binds to KIR3DS 1.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators causes a decrease in activation of one or more of: PD-1, TGF-beta receptor, TIGIT, CD1, TIM-3, siglec-7, IRP60, tactive, IL1R8, NKG2A/KLRD1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1 and LAG-2. In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of PD-1 is an antagonistic antibody that specifically binds to PD-1, soluble PD-L1, or an antibody that specifically binds to PD-L1. In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of the TGF- β receptor is a soluble TGF- β receptor, an antibody that specifically binds to TGF- β, or an antagonistic antibody that specifically binds to TGF- β receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced TIGIT activation is an antagonistic antibody that specifically binds to TIGIT, a soluble TIGIT, or an antibody that specifically binds to TIGIT ligand.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of CD1 is an antagonistic antibody that specifically binds to CD1, soluble CD1, or an antibody that specifically binds to a CD1 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of TIM-3 is an antagonistic antibody that specifically binds to TIM-3, soluble TIM-3, or an antibody that specifically binds to a TIM-3 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of Siglec-7 is an antagonistic antibody that specifically binds to Siglec-7, or an antibody that specifically binds to a Siglec-7 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced IRP60 activation is an antagonistic antibody that specifically binds to IRP60, or an antibody that specifically binds to an IRP60 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of tactive is an antagonistic antibody that specifically binds to tactive, or an antibody that specifically binds to a tactive ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of IL1R8 is an antagonistic antibody that specifically binds to IL1R8, or an antibody that specifically binds to an IL1R8 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of NKG2A/KLRD1 is an antagonistic antibody that specifically binds to NKG2A/KLRD1, or an antibody that specifically binds to a NKG2A/KLRD1 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced KIR2DL1 activation is an antagonistic antibody that specifically binds to KIR2DL1, or an antibody that specifically binds to a KIR2DL1 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced KIR2DL2/3 activation is an antagonistic antibody that specifically binds to KIR2DL2/3, or an antibody that specifically binds to KIR2DL2/3 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced KIR2DL5 activation is an antagonistic antibody that specifically binds to KIR2DL5, or an antibody that specifically binds to a KIR2DL5 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced KIR3DL1 activation is an antagonistic antibody that specifically binds to KIR3DL1, or an antibody that specifically binds to KIR3DL1 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced KIR3DL2 activation is an antagonistic antibody that specifically binds to KIR3DL2, or an antibody that specifically binds to a KIR3DL2 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced activation of ILT2/LIR-1 is an antagonistic antibody that specifically binds to ILT2/LIR-1, or an antibody that specifically binds to an ILT2/LIR-1 ligand.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that cause reduced LAG-2 activation is an antagonistic antibody that specifically binds to LAG-2, or an antibody that specifically binds to LAG-2 ligand.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators is a single chain chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a second target binding domain. In some embodiments of any of the methods described herein, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain. In some embodiments of any of the methods described herein, the soluble tissue factor domain and the second target binding domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain. In some embodiments of any of the methods described herein, the second target binding domain and the soluble tissue factor domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the second target binding domain and the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each an antigen binding domain. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands for TGF-beta receptor II (TGF-beta RII) a ligand for TGF-beta RIII, a ligand for DNAM1, a ligand for NKp46, a ligand for NKp44, a ligand for NKG2D, a ligand for NKp30, a ligand for scMHCI, a ligand for scTCR, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, and a receptor for CD 122.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein. In some embodiments of any one of the methods described herein, the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the methods described herein, the soluble interleukin or cytokine receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble receptor for TNF alpha, soluble receptor for IL-4, or soluble receptor for IL-10.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID No. 93. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID No. 93. In some embodiments of any one of the methods described herein, the soluble human tissue factor domain does not include one or more of the following: lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein; arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

In some embodiments of any one of the methods described herein, the soluble human tissue factor domain does not include any of the following: lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein; arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is not capable of binding factor vila. In some embodiments of any of the methods described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any one of the methods described herein, the single-chain chimeric polypeptide does not stimulate blood clotting in the mammal. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises one or more additional target binding domains located at its N-terminus and/or C-terminus.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus. In some embodiments of any of the methods described herein, the one or more additional target binding domains directly adjoin the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide comprises one or more additional target binding domains located at its C-terminus. In some embodiments of any of the methods described herein, one of the one or more additional target binding domains directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus and C-terminus. In some embodiments of any of the methods described herein, one or more additional antigen binding domains located at the N-terminus directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains located at the N-terminus and the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments of any of the methods described herein, one of the one or more additional antigen binding domains located at the C-terminus directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains located at the C-terminus and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to different antigens.

In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, TGF-beta receptor II (TGF-. Beta.RII) ligand, TGF-. Beta.RIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, PDGF-DD receptor, receptor (SCF-DD receptor), receptor (receptor 3, MICP receptor) receptor of MIC-3, receptor-receptor (CD-B receptor) 122, receptor-3 of MIC-cell-like receptor (receptor 3, receptor-A of MIC-receptor-3.

In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine protein. In some embodiments of any one of the methods described herein, the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine receptor. In some embodiments of any of the methods described herein, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble receptor for TNF alpha, soluble receptor for IL-4, or soluble receptor for IL-10.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) A second domain in a pair of affinity domains, and (ii) a second target binding domain, wherein the first chimeric polypeptide associates with the second chimeric polypeptide via binding of the first domain in the pair of affinity domains to the second domain.

In some embodiments of any of the methods described herein, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the soluble tissue factor domain and the first domain in the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains. In some embodiments of any of the methods described herein, the second domain in a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each an antigen binding domain. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands for TGF-beta receptor II (TGF-beta RII) a ligand for TGF-beta RIII, a ligand for DNAM1, a ligand for NKp46, a ligand for NKp44, a ligand for NKG2D, a ligand for NKp30, a ligand for scMHCI, a ligand for scTCR, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, and a receptor for CD 122.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein. In some embodiments of any one of the methods described herein, the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the methods described herein, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble receptor for TNF alpha, soluble receptor for IL-4, or soluble receptor for IL-10.

In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more additional antigen binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more additional antigen binding domains, and/or a linker sequence between at least one of the one or more additional antigen binding domains and the first domain in the pair of affinity domains.

In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains located at the N-terminus and/or the C-terminus of the first chimeric polypeptide. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains directly abuts the first domain in the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain in the pair of affinity domains. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains directly adjoins the first target binding domain. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain.

In some embodiments of any of the methods described herein, at least one of the one or more additional target binding domains is disposed N-terminal and/or C-terminal to the first chimeric polypeptide, and at least one of the one or more additional target binding domains is located between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed N-terminally directly abuts the first target binding domain or the first domain in the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed at the C-terminus directly abuts the first target binding domain or the first domain in the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains. In some embodiments of any of the methods described herein, at least one of the one or more additional target binding domains located between the soluble tissue factor domain and the first domain of the pair of affinity domains directly adjoins the soluble tissue factor domain and/or the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence disposed between (i) the soluble tissue factor domain and at least one of the one or more additional target binding domains, the target binding domain being located between the soluble tissue factor domain and the first domain of the pair of affinity domains; and/or (ii) between a first domain of a pair of affinity domains and at least one of the one or more additional target binding domains, the target binding domain being located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises one or more additional target binding domains located at the N-terminus and/or C-terminus of the second chimeric polypeptide. In some embodiments of any of the methods described herein, in the second chimeric polypeptide, at least one of the one or more additional target binding domains directly adjoins the second domain in the pair of affinity domains. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the second domain in the pair of affinity domains. In some embodiments of any of the methods described herein, in the second chimeric polypeptide, at least one of the one or more additional target binding domains directly adjoins the second target binding domain. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to different antigens. In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv.

In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, TGF-beta receptor II (TGF-. Beta.RII) ligand, TGF-. Beta.RIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, PDGF-DD receptor, receptor (SCF-DD receptor), receptor (receptor 3, MICP receptor) receptor of MIC-3, receptor-receptor (CD-B receptor) 122, receptor-3 of MIC-cell-like receptor (receptor 3, receptor-A of MIC-receptor-3.

In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine protein. In some embodiments of any one of the methods described herein, the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine receptor. In some embodiments of any of the methods described herein, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble receptor for TNF alpha, soluble receptor for IL-4, or soluble receptor for IL-10.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID No. 93. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID No. 93.

In some embodiments of any one of the methods described herein, the soluble human tissue factor domain does not include one or more of the following: lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein; arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

In some embodiments of any one of the methods described herein, the soluble human tissue factor domain does not include any of the following: lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein; arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is not capable of binding to factor vila. In some embodiments of any of the methods described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any one of the methods described herein, the multi-chain chimeric polypeptide does not stimulate blood clotting in the mammal. In some embodiments of any of the methods described herein, the pair of affinity domains is the sushi domain and soluble IL-15 from the human IL-15 receptor alpha chain (IL-15 ra). In some embodiments of any of the methods described herein, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments of any one of the methods described herein, the human IL-15 ra is mature full length IL-15 ra.

In some embodiments of any one of the methods described herein, the pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, and SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

In some embodiments of any one of the methods described herein, at least one of the one or more NK cell activators is a multi-chain chimeric polypeptide comprising: (a) First and second chimeric polypeptides, each of which comprises: (i) a first target binding domain, (ii) an Fc domain, and (iii) a first domain of a pair of affinity domains; and (b) third and fourth chimeric polypeptides, each comprising: (i) A second domain in a pair of affinity domains and (ii) a second target binding domain, wherein the first and second chimeric polypeptides associate with the third and fourth chimeric polypeptides via binding of the first domain and the second domain in the pair of affinity domains, and the first and second chimeric polypeptides associate via their Fc domains.

In some embodiments of any of the methods described herein, the first target binding domain and the Fc domain in the first and second chimeric polypeptides are directly adjacent to each other in the first and second chimeric polypeptides. In some embodiments of any of the methods described herein, the first and second chimeric polypeptides further comprise a linker sequence between the first target binding domain and the Fc domain in the first and second chimeric polypeptides. In some embodiments of any of the methods described herein, the Fc domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first and second chimeric polypeptides. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the Fc domain in the first and second chimeric polypeptides and the first domain in the pair of affinity domains.

In some embodiments of any of the methods described herein, the second domain in a pair of affinity domains and the second target binding domain are directly adjacent to each other in the third and fourth chimeric polypeptides. In some embodiments of any of the methods described herein, the third and fourth chimeric polypeptides further comprise a linker sequence between the second domain in the pair of affinity domains in the third and fourth chimeric polypeptides and the second target binding domain.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens. In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each an antigen binding domain. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv or a single domain antibody.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands for TGF-beta receptor II (TGF-beta RII) a ligand for TGF-beta RIII, a ligand for DNAM1, a ligand for NKp46, a ligand for NKp44, a ligand for NKG2D, a ligand for NKp30, a ligand for scMHCI, a ligand for scTCR, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, and a receptor for CD 122.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein. In some embodiments of any one of the methods described herein, the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the methods described herein, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble receptor for TNF alpha, soluble receptor for IL-4, or soluble receptor for IL-10.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain that does not stimulate blood clotting. In some embodiments of any of the methods described herein, the soluble tissue factor domain comprises or consists of a sequence of wild-type soluble human tissue factor.

Provided herein are methods of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Also provided herein are methods of reducing accumulation of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Also provided herein are methods of reducing the level of a marker of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Also provided herein are methods of reducing the activity of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Also provided herein are methods of reducing the level and/or activity of one or more SASP factors derived from naturally occurring and/or therapeutically induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

In some embodiments, the subject has been previously diagnosed or identified as having an aging-related disease or inflammatory disease. In some embodiments, the aging-related disorder is inflammatory aging-related.

In some embodiments, the aging-related disorder is selected from the group consisting of: alzheimer's disease, aneurysms, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, disc degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, failure of kidney transplantation, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, parkinson's disease, age-related loss of elasticity of lung tissue, age-related macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

In some embodiments, the aging-related disorder is a cancer selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

In some embodiments, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, crohn's disease, multiple sclerosis, guillain barre syndrome, psoriasis, grave's disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders and cognitive disorders associated with cancer treatment.

In some embodiments, the treatment-induced senescent cells are chemotherapy-induced senescent cells. In some embodiments, administration of one or more common gamma chain family cytokine receptor activators results in a reduction in the number of naturally occurring senescent cells and/or treatment-induced senescent cells in the target tissue of the subject. In some embodiments, the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, vasculature, skeletal tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

In some embodiments, at least one of the one or more common gamma chain family cytokine receptor activators is a complex of a common gamma chain family cytokine or a functional fragment thereof and an antibody or antibody fragment that specifically binds to the common gamma chain family cytokine or a functional fragment thereof.

In some embodiments, at least one of the one or more common gamma chain family cytokine receptor activators is a single chain chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain; (iii) A second target binding domain, wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine, an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common gamma chain family cytokine receptor, or an antigen binding domain that specifically binds to a common gamma chain family cytokine.

In some embodiments, one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine. In some embodiments, the soluble common gamma chain family cytokine is selected from the group consisting of: soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15 and soluble IL-21. In some embodiments, one or both of the first target binding domain and the second target binding domain comprises an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor. In some embodiments, the common gamma chain family cytokine receptor is a receptor for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments, the agonistic antigen binding domain is a scFv, VHH, or VNAR.

In some embodiments, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain. In some embodiments, the soluble tissue factor domain and the second target binding domain are directly adjacent to each other. In some embodiments, the single chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain. In some embodiments, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments, the first target binding domain and the second target binding domain comprise the same amino acid sequence. In some embodiments, the first target binding domain and the second target binding domain specifically bind to different antigens.

In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93. In some embodiments, the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and/or C-terminus.

In some embodiments, at least one of the one or more common gamma chain family cytokine receptor activators is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding polypeptide domain; (ii) a soluble tissue factor domain; (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; (ii) A second target binding domain, wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine, an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common gamma chain family cytokine receptor, or an antigen binding domain that specifically binds to a common gamma chain family cytokine.

In some embodiments, one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine. In some embodiments, the soluble common gamma chain family cytokine is selected from the group consisting of: soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15 and soluble IL-21. In some embodiments, one or both of the first target binding domain and the second target binding domain comprises an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor. In some embodiments, the common gamma chain family cytokine receptor is a receptor for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments, the agonistic antigen binding domain is a scFv, VHH, or VNAR.

In some embodiments, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains. In some embodiments, the second domain and the second target binding domain of a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

In some embodiments, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments, the first target binding domain and the second target binding domain specifically bind to different antigens. In some embodiments, the first chimeric polypeptide further comprises one or more additional target binding domains. In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains.

In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93. In some embodiments, the pair of affinity domains is a sushi domain from the alpha chain of the human IL-15 receptor (IL 15Rα) and soluble IL-15. In some embodiments, the pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

In some embodiments, the first domain or the second domain of a pair of affinity domains is a soluble common gamma chain family cytokine or an antigen binding domain that specifically binds to a common gamma chain family cytokine receptor. In some embodiments, at least one of the one or more common gamma chain family cytokine receptor activators is a soluble IL-15 or IL-15 agonist. In some embodiments, soluble IL-15 is at least 90% identical to SEQ ID NO. 82. In some embodiments, IL-15 agonists include a complex of IL-15 and all or part of the soluble IL-15 receptor (IL-15R). In some embodiments, the partially soluble IL-15R is a portion of IL-15Rα. In some embodiments, the partially soluble IL-15Rα is the sushi domain of IL-15Rα. In some embodiments, the IL-15 agonist further comprises an Fc domain. In some embodiments, an IL-15 agonist comprises a fusion protein comprising IL-15 and a sushi domain from IL-15Rα. In some embodiments, one of the one or more common gamma chain family cytokine receptor activators is a soluble IL-2 or IL-2 agonist. In some embodiments, one of the one or more common gamma chain family cytokine receptor activators is an antibody or antigen-binding antibody fragment that specifically binds a common gamma chain family cytokine.

In some embodiments, the method comprises administering one, two, or more doses of one or more common gamma chain family cytokine receptor activators to the subject. In some embodiments, any two consecutive doses of the two or more doses are administered between about 1 week and about one year apart. In some embodiments, any two consecutive doses of the two or more doses are administered between about 1 week and about 6 months apart. In some embodiments, any two consecutive doses of the two or more doses are administered between about 1 week and about 2 months apart. In some embodiments, any two consecutive doses of the two or more doses are administered between about 1 week and about 1 month apart.

In some embodiments, one, two, or more doses are administered by subcutaneous administration. In some embodiments, the two or more doses are administered by intramuscular injection. In some embodiments, two or more doses are administered over a period of about 1 year to about 60 years. In some embodiments, two or more doses are administered over a period of about 1 year to about 50 years. In some embodiments, two or more doses are administered over a period of about 1 year to about 40 years. In some embodiments, two or more doses are administered over a period of about 1 year to about 30 years. In some embodiments, two or more doses are administered over a period of about 1 year to about 20 years. In some embodiments, two or more doses are administered over a period of about 1 year to about 10 years.

In some embodiments, each of the two or more doses is administered at a dose of about 0.01mg/kg of each common gamma chain family cytokine receptor activator to about 10mg/kg of each common gamma chain family cytokine receptor activator. In some embodiments, each of the two or more doses is administered at a dose of about 0.02mg/kg of each common gamma chain family cytokine receptor activator to about 5mg/kg of each common gamma chain family cytokine receptor activator.

In some embodiments, the first dose of one or more common gamma chain family cytokine receptor activators begins when the subject reaches at least 30 years of age. In some embodiments, the first dose of one or more common gamma chain family cytokine receptor activators begins when the subject reaches at least 40 years of age. In some embodiments, the first dose of one or more common gamma chain family cytokine receptor activators begins when the subject reaches at least 50 years of age. In some embodiments, the first dose of one or more common gamma chain family cytokine receptor activators begins when the subject reaches at least 60 years of age.

In some embodiments, the subject is not diagnosed or identified as having an aging-related disease or inflammatory disease. In some embodiments, the subject has not been previously treated with a chemotherapeutic agent. In some embodiments, the subject has not been previously treated with a therapeutic agent that induces cellular senescence. In some embodiments, the method further comprises administering to the subject at least one or more agents that result in reduced TGF- β receptor activation. In some embodiments, the agent that results in reduced activation of the TGF- β receptor is a soluble TGF- β receptor, an extracellular domain of a TGF- β receptor, an antibody that specifically binds to TGF- β, an antagonistic antibody that binds to a TGF- β receptor, an agent that binds to LAP, or an agent that binds to a TGF- β/LAP complex. In some embodiments, one or more agents that result in reduced TGF- β activation attenuate activation of TGF- β receptors by binding to LAP or TGF- β/LAP complex.

In some embodiments, the soluble human tissue factor domain does not trigger blood clotting. In some embodiments, the method further comprises administering an additional therapeutic agent selected from the group consisting of: combinations of agents, such as checkpoint inhibitors, chemotherapeutic agents and therapeutic antibodies.

In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide is stable in human serum at 37 ℃ for at least 10 days. In some embodiments of any of the methods described herein, the multi-chain chimeric polypeptide is stable in human serum at 37 ℃ for at least 10 days. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide does not have significant clotting activity. In some embodiments of any of the methods described herein, the multi-chain chimeric polypeptide does not have significant clotting activity.

In some embodiments of any one of the methods described herein, the method regenerates the aged immune cells in the subject. In some embodiments of any of the methods described herein, regeneration of the senescent immune cells results in a reduction in the number of diseased cells or infectious agents in the subject. In some embodiments of any of the methods described herein, the aged immune cells comprise one or more of aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils, aged basophils, aged eosinophils, aged Kupffer cells, and aged microglia. In some embodiments of any of the methods described herein, the diseased cells include cancer cells, virus-infected cells, and intracellular bacteria-infected cells. In some embodiments of any of the methods described herein, the infectious agent comprises a virus, a bacterium, a fungus, and a parasite.

As used herein, the term "chimeric" refers to polypeptides that include amino acid sequences (e.g., domains) that were originally derived from two different sources (e.g., two naturally occurring different proteins, e.g., from the same or different species). For example, a chimeric polypeptide may include domains from at least two different naturally occurring human proteins. In some embodiments, a chimeric polypeptide may include a domain that is a synthetic sequence (e.g., an scFv) and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide may include at least two different domains that are synthetic sequences (e.g., two different scFv).

An "activated NK cell" is an NK cell that exhibits elevated expression levels of two or more (e.g., three, four, five, or six) of CD25, CD69, MTOR-C1, SREBP, IFN- γ, and granzyme (e.g., granzyme B), for example, as compared to a resting NK cell. Described herein are exemplary methods for identifying the expression levels of CD25, CD69, MTOR-C1, SREBP, IFN- γ, and granzyme (e.g., granzyme B).

A "resting NK cell" is an NK cell whose expression is reduced in two or more (e.g. three, four, five or six) of CD25, CD69, MTOR-C1, SREBP, IFN-gamma and granzyme (e.g. granzyme B) compared to an activated NK cell, for example.

An "NK cell activator" is an agent that induces or promotes (alone or in combination with other NK cell activators) the development of resting NK cells into activated NK cells. Non-limiting examples and aspects of NK cell activators are described herein.

An "antigen binding domain" is one or more protein domains capable of specifically binding to one or more different antigens (e.g., formed from amino acids from a single polypeptide or from amino acids from two or more polypeptides (e.g., the same or different polypeptides)). In some embodiments, the antigen binding domain may bind to an antigen or epitope with similar specificity and affinity as a naturally occurring antibody. In some embodiments, the antigen binding domain may be an antibody or fragment thereof. In some embodiments, the antigen binding domain may comprise a surrogate scaffold. Non-limiting examples of antigen binding domains are described herein. Additional examples of antigen binding domains are known in the art.

"soluble tissue factor domain" refers to a polypeptide that has at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) to a segment of a wild-type mammalian tissue factor protein (e.g., a wild-type human tissue factor protein) that lacks a transmembrane domain and an intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

The term "soluble interleukin protein" is used herein to refer to a mature and secreted interleukin protein or biologically active fragment thereof. In some embodiments, the soluble interleukin protein may include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature and secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein), and retains its biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

The term "soluble cytokine protein" is used herein to refer to a mature and secreted cytokine protein or biologically active fragment thereof. In some embodiments, the soluble cytokine protein can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature and secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein), and retains its biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

The term "soluble interleukin receptor" is used herein in its broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) capable of binding one or more of its natural ligands (e.g., under physiological conditions, e.g., at room temperature, in phosphate buffered saline). For example, a soluble interleukin receptor may include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type interleukin receptor, and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally also lacks its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

The term "soluble cytokine receptor" is used herein in its broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) capable of binding one or more of its natural ligands (e.g., under physiological conditions, e.g., at room temperature, in phosphate buffered saline). For example, a soluble cytokine receptor may include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type cytokine receptor, and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally also lacks its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen binding domains that specifically bind to an antigen or epitope. Antibodies include, in particular, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. One example of an antigen binding domain is one formed from VH-VL dimers. Additional embodiments of antibodies are described herein. Additional examples of antibodies are known in the art.

"affinity" refers to the strength of the sum of non-covalent interactions between an antigen binding site and its binding partner (e.g., antigen or epitope). As used herein, unless otherwise indicated, "affinity" refers to an inherent binding affinity that reflects a 1:1 interaction between an antigen binding domain and a member of an antigen or epitope. The affinity of a molecule X for its partner Y can be expressed by the dissociation equilibrium constant (KD). The kinetic components contributing to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including the methods described herein. Affinity can be Such as using Surface Plasmon Resonance (SPR) techniques (e.g.,) Or interferometry of biological layers (e.g.,) To determine. Additional methods of determining the affinity for an antigen binding domain and its corresponding antigen or epitope are known in the art.

As used herein, "single chain polypeptide" refers to a single protein chain.

As used herein, a "multi-chain polypeptide" refers to a polypeptide comprising two or more (e.g., three, four, five, six, seven, eight, nine, or ten) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), wherein the two or more protein chains associate via non-covalent bonds to form a quaternary structure.

The term "a pair of affinity domains" is in less than 1x10 ^-7 M (e.g. less than 1x10 ^-8 M is less than 1x10 ^-9 M is less than 1x10 ^-10 M or less than 1x10 ^-11 K of M) _D Two different protein domains that specifically bind to each other. In some embodiments, the pair of affinity domains can be a pair of naturally occurring proteins. In some embodiments, a pair of affinity domains can be a pair of synthetic proteins. Non-limiting examples of a pair of affinity domains are described herein.

The term "epitope" refers to a portion of an antigen that specifically binds to an antigen binding domain. Epitopes can for example consist of surface accessible amino acid residues and/or sugar side chains and can have specific three-dimensional structural features as well as specific charge features. Conformational and non-conformational epitopes differ in that binding to the former, but not to the latter, may be lost in the presence of denaturing solvents. Epitopes may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. Methods for identifying epitopes that bind to antigen binding domains are known in the art.

The term "treating" refers to ameliorating at least one condition of a condition. In some embodiments, the disorder treated is cancer, and ameliorating at least one disorder of the cancer includes reducing abnormal proliferation, gene expression, signaling, translation, and/or secretion of factors. Generally, a method of treatment comprises administering to a subject in need or having been determined to be in need of such treatment a therapeutically effective amount of a composition, thereby reducing at least one disorder of the disorder.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Drawings

FIGS. 1A-1B show the results of immunostimulation of an exemplary multi-chain polypeptide in a C57BL/6 mouse. Fig. 1A shows spleen weights of mice treated with increasing doses of exemplary multi-chain polypeptides compared to mice treated with control solutions. Fig. 1B shows the percentage of immune cell types present in the spleen of mice treated with increasing doses of the exemplary multi-chain polypeptide compared to control solution treated mice.

FIGS. 2A-2B show the duration of immunostimulation of an exemplary multi-chain polypeptide in a C57BL/6 mouse. FIG. 2A shows the weight of spleen of mice treated with 3mg/kg of an exemplary multi-chain polypeptide over a 92 hour period. FIG. 2B shows the percentage of immune cell types present in the spleen of mice treated with 3mg/kg of an exemplary multi-chain polypeptide over a 92 hour period.

FIGS. 3A-3B show immunity induced by exemplary multi-chain polypeptidesExpression of Ki67 and granzyme B in epidemic cells. FIG. 3A shows CD4 at various time points after treatment with a multi-chain polypeptide ⁺ T cells, CD8 ⁺ T cells, natural Killer (NK) cells and CD19 ⁺ Expression of Ki67 in B cells. FIG. 3B shows CD4 at various time points after treatment with a multi-chain polypeptide ⁺ T cells, CD8 ⁺ T cells, natural Killer (NK) cells and CD19 ⁺ Expression of granzyme B by B cells.

Fig. 4 shows tumor-inhibiting effects of splenocytes prepared from exemplary multi-chain polypeptide treated mice at various time points after treatment.

FIGS. 5A-5B show B6.129P2-ApoE fed control, high fat and untreated ^tm1Unc CD4 in blood of/J mice (purchased from Jackson laboratories (Jackson Laboratory)) and mice fed high fat diet and treated with TGFRt15-TGFRs, 2t2 or 21t15-TGFRs ⁺ T cells, CD8 ⁺ T cells, natural Killer (NK) cells and CD19 ⁺ Percentage of B cells and proliferation rate. Fig. 5A shows the percentage of different cell types in each control and experimental group. Fig. 5B shows proliferation rates of different cell types in each control and experimental group.

Fig. 6A-6E show exemplary physical appearance of mice fed control or high fat diet and untreated or treated with TGFRt15-TGFRs, 2t2 or 21t 15-TGFRs.

Figure 7 shows the fasting body weight of mice fed control or high fat diet and untreated or treated with TGFRt15-TGFRs, 2t2 or 21t 15-TGFRs.

FIG. 8 shows fasting blood glucose levels in mice fed control or high fat diet and untreated or treated with TGFRt15-TGFRs, 2t2 or 21t 15-TGFRs.

FIGS. 9A-9F show chemotherapy-induced expression of senescent B16F10 cells and senescent genes. FIG. 9A shows chemotherapy-induced senescent B16F10 cells visualized using SA β -gal staining. FIGS. 9B-9F show p21 in chemotherapy-induced senescent B16F10 cells ^CIP1 IL6, DPP4, RATE1E and ULBP1 are expressed over time.

FIGS. 10A-10F show chemotherapy-induced colony formation of senescent B16F10 cells and expression of stem cell markers. Figure 10A shows chemotherapy-induced colony formation of senescent B16F10 cells. Figures 10B and 10C show chemotherapy-induced expression of Oct4 mRNA and Notch4 mRNA of senescent B16F10 cells compared to control B16F10 cells. Figures 10D-10F show the percentage of chemotherapy-induced aging B16F10 cells that were biscationic for two of the three stem cell markers (including CD44, CD24, and CD 133).

FIGS. 11A-11C show migration and invasion characteristics of senescent B16F10 cells induced by chemotherapy. FIG. 11A shows the results of a migration assay comparing chemotherapy-induced senescent cells with stem cell characteristics (B16F 10-SNC-CSC) with control B16F10 cells. Figures 11B and 11C show the results of an invasive analysis comparing chemotherapy-induced senescent cells with stem cell characteristics (B16F 10-SNC-CSC) with control B16F10 cells.

FIGS. 12A and 12B show the cytotoxicity of NK cells expanded in vitro and of chemotherapy-induced senescent cells (B16F 10-SNC-CSC) with stem cell properties or control B16F10 cells. FIG. 12A shows an exemplary process schematic for obtaining NK cells expanded in vitro. FIG. 12B shows cytotoxicity of expanded NK cells against chemotherapy-induced senescent cells (B16F 10-SNC-CSC) with stem cell properties or control B16F10 cells.

Fig. 13A-13C show the results of combination treatment using the mouse melanoma model. Fig. 13A shows an exemplary schematic for treating melanoma in a mouse model. Figures 13B and 13C show the change in tumor volume over time for combination treatments including TGFRt15-TGFRs compared to chemotherapy or TA99 treatment alone.

FIG. 14 shows induction of senescence in the human pancreatic tumor cell line SW1990 and expression of CD44 and CD24 in senescent SW1990 cells compared to control SW1990 cells.

FIG. 15 shows expression of senescence markers in chemotherapy-induced senescent SW1990 cells.

FIG. 16 shows cytotoxicity of in vitro activated human NK cells against chemotherapy-induced aging SW1990 cells or control SW1990 cells.

FIG. 17 shows a schematic representation of an exemplary IL-12/IL-15RαSu DNA construct.

FIG. 18 shows a schematic representation of an exemplary IL-18/TF/IL-15 DNA construct.

FIG. 19 shows a schematic representation of the interaction between an exemplary IL-12/IL-15RαSu and an IL-18/TF/IL-15 DNA construct.

FIG. 20 shows a schematic representation of the interaction between an exemplary IL-12/IL-15RαSu and an IL-18/TF/IL-15 fusion protein to produce an IL-18/TF/IL-15:IL-12/IL-15RαSu complex (18 t15-12 s).

FIG. 21 shows the elution of anti-TF antibody affinity column purification for 18t15-12 s.

FIG. 22 shows exemplary chromatographic profiles of anti-TF Ab/SEC purified 18t15-12s proteins after elution on an analytical size exclusion column, demonstrating separation of the polyprotein monomer 18t15-12s complex from protein aggregates.

FIG. 23 shows an example of 4-12% SDS-PAGE of 18t15-12s complexes after disulfide reduction. Lane 1: a SeeBlue Plus2 marker; lane 2: anti-TF Ab purified 18t15-12s (0.5 μg); lane 3: anti-TF Ab purified 18t15-12s (1 μg).

FIG. 24 shows SDS PAGE analysis of deglycosylated and non-deglycosylated 18t15-12 s. Lane 1: anti-TF Ab purified 18t15-12s (0.5 μg), not deglycosylated; lane 2: anti-TF Ab purified 18t15-12s (1 μg), not deglycosylated; lane 3:18t15-12s (1 μg), deglycosylation; lane 4: mark12 was not a dyed marker.

FIG. 25 shows a sandwich ELISA for the 18t15-12s complex comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 detection antibody (BAF 219).

FIG. 26 shows a sandwich ELISA for the 18t15-12s complex comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247).

FIG. 27 shows a sandwich ELISA for the 18t15-12s complex comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-18 detection antibody (D045-6).

FIG. 28 shows a sandwich ELISA for the 18t15-12s complex comprising an anti-human tissue factor (I43) capture antibody and an anti-human tissue factor detection antibody.

FIG. 29 shows IL-15 dependent 32D beta cell proliferation mediated by the 18t15-12s complex (open squares) and recombinant IL-15 (black squares).

FIG. 30 shows the bioactivity of IL-18 within the 18t15-12s complex (open squares), where recombinant IL-18 (black squares) and recombinant IL-12 (black circles) served as positive and negative controls, respectively.

FIG. 31 shows the bioactivity of IL-12 within the 18t15-12s complex (open squares), where recombinant IL-12 (black circles) and recombinant IL-18 (open squares) served as positive and negative controls, respectively.

FIGS. 32A and 32B show cell surface expression of CD25 on NK cells induced by the 18t15-12s complex and CD69 expression on cell surfaces of NK cells induced by the 18t15-12s complex.

FIG. 33 shows a flow cytometry pattern of induction of NK cells to express IFN- γ in cells by 18t15-12s complex.

FIG. 34 shows cytotoxicity of human NK cells against K562 cells induced by 18t15-12 s.

FIG. 35 shows a schematic of an exemplary IL-12/IL-15RαSu/αCD16 DNA construct.

FIG. 36 shows a schematic of an exemplary IL-18/TF/IL-15 DNA construct.

FIG. 37 shows a schematic representation of the interaction between an exemplary IL-12/IL-15RαSu/αCD16scFv and an IL-18/TF/IL-15 DNA construct.

FIG. 38 shows a schematic of an exemplary 18t15-12s/αCD16 protein complex.

FIG. 39 shows a sandwich ELISA for the 18t15-12s16 complex comprising an anti-human tissue factor antibody capture antibody and biotinylated anti-human IL-12 (BAF 219) (dark line) or anti-human tissue factor detection antibody (light line).

FIG. 40 shows a schematic diagram of an exemplary TGF-beta RII/IL-15RαSu DNA construct.

FIG. 41 shows a schematic of an exemplary IL-21/TF/IL-15 construct.

FIG. 42 shows a schematic representation of the interaction between exemplary IL-IL-21/TF/IL-15 and TGF-beta RII/IL-15RαSu constructs.

FIG. 43 shows a schematic representation of the interaction between an exemplary TGF-beta RII/IL-15R alpha Su and an IL-21/TF/IL-15 fusion protein to produce an IL-21/TF/IL-15/TGF-beta RII/IL-15R alpha Su complex (21 t 15-TGFRs).

FIG. 44 shows the chromatography of anti-TF antibody affinity column purification elution 21t 15-TGFRs.

FIG. 45 shows an exemplary 21t15-TGFRs size exclusion chromatography showing major protein peaks and high molecular weight peaks.

FIG. 46 shows an example of 4-12% SDS-PAGE of 21t15-TGFRs complexes after disulfide bond reduction. Lane 1: mark12 undyed marker (left hand number indicates molecular weight (kDa)); lane 2:21t15-TGFRs (0.5 μg); lane 3:21t15-TGFRs (1 μg); lane 4:21t15-TGFRs, deglycosylated (1 μg), where MW is the expected size of 53kDa and 39.08 kDa.

FIG. 47 shows a sandwich ELISA for 21t15-TGFRs complex comprising anti-human tissue factor capture antibody and biotinylated anti-human IL-21 detection antibody (13-7218-81, bioLegend).

FIG. 48 shows a sandwich ELISA for 21t15-TGFRs complexes comprising anti-human tissue factor capture antibody and biotinylated anti-human IL-15 detection antibody (BAM 247, andi biosystems).

FIG. 49 shows a sandwich ELISA for the 21t15-TGFRs complex comprising an anti-human tissue factor capture antibody and a biotinylated anti-human TGF beta RII detection antibody (BAF 241, andi organism).

FIG. 50 shows a sandwich ELISA for 21t15-TGFRs complexes comprising an anti-human tissue factor (I43) capture antibody and an anti-human tissue factor detection antibody.

FIG. 51 shows that the 21t15-TGFRs complex (open squares) mediates IL-15 dependent proliferation of 32D beta cells compared to IL-15 (black squares).

FIG. 52 shows the biological activity of the TGFβRII domain within the 21t15-TGFRs complex (open squares). TGF-beta RII/Fc (black squares) served as a positive control.

FIG. 53 shows a flow cytometry plot of CD25 expression on the cell surface of NK cells induced by 21t15-TGFRs complex.

FIG. 54 shows a flow cytometry plot of CD69 expression on the cell surface of NK cells induced by 21t15-TGFRs complex.

FIG. 55 shows a flow cytometry plot of intracellular IFN-gamma expression of 21t15-TGFRs complex induced NK cells.

FIG. 56 shows that 21t15-TGFRs induces cytotoxicity of human NK cells against K562 cells.

FIG. 57 is a schematic diagram of an exemplary αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide.

FIG. 58 is a chromatogram showing elution of an exemplary αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide from an anti-tissue factor affinity column.

FIG. 59 is a chromatogram showing elution of a Superdex200 Increase 10/300 GL gel filtration column loaded with an exemplary αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide.

FIG. 60 is a diagram of sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) of an exemplary αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide purified using an anti-tissue factor affinity column.

FIG. 61 is a graph showing the quantitation of exemplary αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptides by ELISA performed using the method described in example 1. Purified tissue factor was used as a control.

FIG. 62 is a graph showing that exemplary αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptides stimulate CD4 separation from blood of two donors ⁺ Graph of the ability of T cells to express CD 25. Experiments were performed as described in example 2.

FIG. 63 is a graph showing that an exemplary αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide stimulates CD8 separation from blood of two donors ⁺ Graph of the ability of T cells to express CD 25. Experiments were performed as described in example 2.

FIG. 64 is a displayExemplary αcd3scFv/TF/αcd28scFv single chain chimeric polypeptides stimulated CD4 isolation from blood of two donors ⁺ Graph of the ability of T cells to express CD 69. Experiments were performed as described in example 2.

FIG. 65 shows a schematic of an exemplary IL-7/IL-15RαSu DNA construct.

FIG. 66 shows a schematic representation of an exemplary IL-21/TF/IL-15 DNA construct.

FIG. 67 shows a schematic diagram of the interaction between an exemplary IL-7/IL-15RαSu and an IL-21/TF/IL-15 DNA construct.

FIG. 68 shows a schematic of the interaction between an exemplary IL-7/IL-15RαSu and an IL-21/TF/IL-15 fusion protein to produce an IL-21/TF/IL-15:IL-7/IL-15RαSu complex (21 t15-7 s).

FIG. 69 shows a schematic of an exemplary IL-21/IL-15RαSu DNA construct.

FIG. 70 shows a schematic representation of an exemplary IL-7/TF/IL-15 DNA construct.

FIG. 71 shows a schematic of the interaction between an exemplary IL-21/IL-15RαSu and an IL-7/TF/IL-15 DNA construct.

FIG. 72 shows a schematic representation of the interaction between an exemplary IL-21/IL-15RαSu and an IL-7/TF/IL-15 fusion protein to produce an IL-7/TF/IL-15:IL-21/IL-15RαSu complex (7 t15-21 s).

FIG. 73 shows human NK cells isolated from blood of two different donors (2X 10 ⁶ Individual cells/mL) Oxygen Consumption Rate (OCR) in pmoles/min.

FIG. 74 shows human NK cells isolated from blood of two different donors (2X 10 ⁶ Individual cells/mL) extracellular acidification rate (ECAR), in mpH/min.

FIG. 75 shows a schematic of the 7t15-16s21 construct.

FIG. 76 shows another schematic of the 7t15-16s21 construct.

FIGS. 77A and 77B show the binding of 7t15-16s21 to CHO cells expressing human CD16B compared to control proteins.

FIGS. 78A-78C are results from ELISA experiments using antibodies to IL-15, IL-21 and IL-7 to detect 7t15-16s 21.

FIG. 79 shows the results of a 32D beta cell proliferation assay with 7t15-16s21 or recombinant IL-15.

FIG. 80 shows the chromatographic profile of cell culture supernatants containing 7t15-16s21 protein after binding and elution on anti-TF antibody resin.

FIG. 81 shows analytical SEC profiles of 7t15-16s 21.

FIG. 82 shows a schematic representation of the TGFRt15-16s21 construct.

FIG. 83 shows another schematic representation of the TGFRt15-16s21 construct.

FIGS. 84A and 84B show the binding affinities of TGFRT15-16S21 and 7t15-21S to CHO cells expressing human CD 16B. FIG. 84A shows the binding affinity of TGFRT15-16S21 to CHO cells expressing human CD16 b. FIG. 84B shows the binding affinity of 7t15-21s to CHO cells expressing human CD 16B.

FIG. 85 shows the results of TGFRt15-16s21 and TGFR-Fc inhibition of TGF beta 1.

FIG. 86 shows the results of 32D beta cell proliferation assays with TGFRt15-16s21 or recombinant IL-15.

FIGS. 87A-87C show the results of detection of IL-15, IL-21 and TGF-beta RII in TGFRt15-16s21 using ELISA with the corresponding antibodies.

FIG. 88 shows the chromatographic profile of cell culture supernatants containing TGFRt15-16s21 protein after binding and elution on anti-TF antibody resin.

FIG. 89 shows the results of a reduced SDS-PAGE analysis of TGFRt15-16s 21.

FIG. 90 shows a schematic of the 7t15-7s construct.

FIG. 91 shows another schematic of the 7t15-7s construct.

FIG. 92 shows the chromatographic profile of cell culture supernatants containing 7t15-7s protein after binding and elution on anti-TF antibody resin.

FIG. 93 shows the detection of TF, IL-15 and IL-7 in 7t15-7s using ELISA.

Figures 94A and 94B show spleen weights and percentages of immune cell types for mice treated with 7t15-7s and control treated. FIG. 94A showsSpleen weights of 7t15-7s treated mice compared to PBS control group. FIG. 94B shows CD4 in mice treated with 7t15-7s compared to PBS control ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells.

FIG. 95 shows a schematic representation of the TGFRt15-TGFRs construct.

FIG. 96 shows another schematic of the TGFRt15-TGFRs construct.

FIG. 97 shows the results of TGFRt15-TGFRs and TGFR-Fc induced TGF beta 1 inhibition.

FIG. 98 shows the results of a 32D beta cell proliferation assay using TGFRt15-TGFRs or recombinant IL-15.

FIGS. 99A and 99B show the results of detection of IL-15 and TGFβRII in TGFRt15-TGFRs using ELISA using the corresponding antibodies.

FIG. 100 is a line graph showing the chromatographic profile of cell culture supernatants containing TGFRt15-TGFRs protein after binding and elution on anti-TF antibody resin.

FIG. 101 shows analytical SEC profiles of TGFRt15-TGFRs.

FIG. 102 shows TGFRt15-TGFRs before and after deglycosylation using reduced SDS-PAGE analysis.

Figures 103A and 103B show spleen weights and percentages of immune cell types for TGFRt15-TGFRs treated and control treated mice. FIG. 103A shows spleen weights of mice treated with TGFRt15-TGFRs compared to PBS control. FIG. 103B shows CD4 in TGFRt15-TGFRs treated mice compared to PBS control ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells.

Figures 104A and 104B show spleen weight and immune stimulation during 92 hours in mice treated with TGFRt15-TGFRs. Fig. 104A shows spleen weights at 16, 24, 48, 72 and 92 hours after treatment in TGFRt15-TGFRs treated mice. FIG. 104B shows the percentage of immune cells at 16, 24, 48, 72 and 92 hours after treatment in TGFRt15-TGFRs treated mice.

FIGS. 105A and 105B show the expression of Ki67 and granzyme B over a period of time in TGFRt15-TGFRs treated mice.

FIG. 106 shows the enhancement of cytotoxicity of TGFRt15-TGFRs on spleen cells of C57BL/6 mice.

FIG. 107 shows the change in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or chemotherapy in combination with TGFRt15-TGFRs in a mouse model of pancreatic cancer.

FIG. 108 shows cytotoxicity of NK cells isolated from TGFRt15-TGFRs treated mice.

FIG. 109 shows a schematic of 7t15-21s137L (long version) construct.

FIG. 110 shows another schematic of 7t15-21s137L (long version) construct.

FIG. 111 is a graph showing the chromatographic profile of cell culture supernatants containing 7t15-21s137L (long form) protein after binding and elution on anti-TF antibody resin.

FIG. 112 shows analytical SEC profiles of 7t15-21s137L (long panel).

FIG. 113 shows the binding of 7t15-21s137L (short version) to CD137L (4.1 BBL).

FIGS. 114A-114C show the detection of IL-15, IL21 and IL7 in 7t15-21s137L (short version) by ELISA. FIG. 114A shows the detection of IL-15 in 7t15-21s137L (short version) by ELISA. FIG. 114B shows the detection of IL21 in 7t15-21s137L (short version) by ELISA. FIG. 114C shows the detection of IL7 in 7t15-21s137L (short version) by ELISA.

FIG. 115 shows the results of CTLL-2 cell proliferation assays.

FIG. 116 shows the activity of 7t15-1s137L (short version) in promoting proliferation of B9 cells containing IL 21R.

FIG. 117 shows a schematic representation of the 7t15-TGFRs construct.

FIG. 118 shows another schematic of the 7t15-TGFRs construct.

FIG. 119 shows the results of TGF-beta 1 inhibition by 7t15-TGFRs and TGFR-Fc.

FIGS. 120A-120C show ELISA detection of IL-15, TGFβRII and IL-7 in 7t15-TGFRs.

FIG. 121 shows the results of 32D beta cell proliferation assays performed with 7t15-TGFRs or recombinant IL-15.

FIG. 122 is a line graph showing the chromatographic profile of cell culture supernatants containing 7t15-TGFRs protein after binding and elution on anti-TF antibody resin.

FIG. 123 shows 7t15-TGFRs before and after deglycosylation using reduced SDS-PAGE analysis.

FIG. 124 shows ELISA detection of IL-7, IL-15 and TGF-beta RII in 7t15-TGFRs proteins.

Figures 125A and 125B show spleen weights and percentages of immune cell types for mice treated with 7t15-TGFRs and control. FIG. 125A shows spleen weights of mice treated with different doses of 7t15-TGFRs compared to PBS control. FIG. 125B shows CD4 in mice treated with different doses of 7t15-TGFRs compared to PBS control ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells.

FIGS. 126A and 126B show the upregulation of CD44 expression by CD4+ and CD8+ T cells by 7T15-TGFRs in C57BL/6 mice.

FIGS. 127A and 127B show the up-regulation of Ki67 expression and granzyme B expression by 7T15-TGFRs on CD8+ T cells and NK cells in C57BL/6 mice.

FIG. 128 shows that 7t15-TGFRs enhances cytotoxicity of spleen cells of C57BL/6 mice.

FIG. 129 shows a schematic of the TGFRt15-21s137L construct.

FIG. 130 shows another schematic representation of the TGFRt15-21s137L construct.

FIG. 131 is a graph showing the chromatographic profile of cell culture supernatants containing TGFRt15-21s137L protein after binding and elution on anti-TF antibody resin.

FIG. 132 shows a schematic representation of the TGFRt15-TGFRs21 construct.

FIG. 133 shows another schematic of the TGFRt15-TGFRs21 construct.

FIG. 134 is a line graph showing the chromatographic profile of cell culture supernatants containing TGFRt15-TGFRs21 protein after binding and elution on anti-TF antibody resin.

FIG. 135 shows TGFRt15-TGFRs21 before and after deglycosylation by reduced SDS-PAGE analysis.

FIGS. 136A and 136B show the detection of components in TGFRt15-TGFRs21 using ELISA.

FIGS. 137A and 137B show CD4 present in spleens of control-treated and TGFRt15-TGFRs 21-treated mice ⁺ T cells, CD8 ⁺ Percentage and proliferation of T cells and Natural Killer (NK) cells.

FIG. 138 shows the upregulation of granzyme B expression in spleen cells of TGFRt15-TGFRs21 treated mice.

FIG. 139 shows that TGFRt15-TGFRs21 enhances the cytotoxicity of spleen cells of C57BL/6 mice.

FIG. 140 shows a schematic representation of the TGFRt15-TGFRs16 construct.

FIG. 141 shows another schematic representation of the TGFRt15-TGFRs16 construct.

FIG. 142 shows a schematic representation of the TGFRt15-TGFRs137L construct.

FIG. 143 shows another schematic representation of the TGFRt15-TGFRs137L construct.

FIG. 144 is a schematic diagram of an exemplary 2t2 single chain chimeric polypeptide.

FIG. 145 shows a comparison of IL-2 activity in 2t2 with recombinant IL-2 as measured using a 32D beta cell proliferation assay.

FIG. 146 shows a comparison of IL-2 activity in 2t2 with recombinant IL-2 as measured using CTLL-2 cell proliferation assay.

FIG. 147 shows ApoE fed standard diet (chow) or high fat diet (diet) and treated with PBS control (untreated) or 2t2 ^-/- Fasting blood glucose levels in mice.

FIG. 148 shows ApoE fed standard or high fat diet and treated with PBS control (untreated) or 2t2 ^-/- CD4 in blood lymphocytes of mice ⁺ CD25 ⁺ FoxP3 ⁺ T regulates cell rate.

FIG. 149 is a line graph showing the chromatographic profile of cell culture supernatants containing 2t2 protein after binding and elution on anti-TF antibody resin.

Graph 150 shows an analytical SEC profile for 2t 2.

FIGS. 151A and 151B show SDS-PAGE analysis of 2t2 before and after deglycosylation. FIG. 151A shows a reduced SDS-PAGE analysis of 2t2 prior to deglycosylation. FIG. 151B shows a reduced SDS-PAGE analysis of 2t2 after deglycosylation.

FIGS. 152A and 152B show the results of immunostimulation of C57BL/6 mice using 2t 2. Fig. 152A shows spleen weight after treatment with 2t 2. Figure 152B shows the percentage of immune cell types after 2t2 treatment.

FIG. 153 shows CD4 in mice treated with 2t2 ⁺ Up-regulation of CD25 expression by T cells.

FIG. 154 shows the pharmacokinetics of 2t2 in C57BL/6 mice.

FIGS. 155A and 155B show that 2t2 is alleviating ApoE ^-/- Effects on high fat-induced atherosclerotic plaque formation in mice. FIG. 155A shows ApoE fed standard or high fat diet and treated with PBS control or 2t2 ^-/- Representative view of atherosclerotic plaques in mice. Fig. 155B shows the results of quantitative analysis of atherosclerotic plaques for each group.

Fig. 156 shows fasting glucose levels in 2t2 treated mice compared to control treated mice.

FIG. 157 shows CD4 in blood lymphocytes of 2t2 treated mice and control treated mice ⁺ CD25 ⁺ FoxP3 ⁺ Tregs percentage.

FIG. 158 is a schematic representation of an exemplary 15t15 single chain chimeric polypeptide.

FIG. 159 shows a comparison of IL-15 activity in 15t15 with recombinant IL-15 as measured by 32D beta cell proliferation assay.

FIG. 160 is a line graph showing the chromatographic profile of cell culture supernatants containing 15t15 protein after binding and elution on anti-TF antibody resin.

FIGS. 161A and 161B show reduced SDS-PAGE analysis of 15t15 before and after deglycosylation. FIG. 161A shows a reduced SDS-PAGE analysis of 15t15 prior to deglycosylation. FIG. 161B shows a reduced SDS-PAGE analysis of 15t15 after deglycosylation.

FIGS. 162A and 162B are a set of histograms (FIG. 162A) and a set of histograms (FIG. 162B) showing the change in NK cell surface phenotype after 18t15-12s, 18t15-12s16 and 7t15-21s+ anti-TF antibody stimulation.

FIG. 163 is a set of graphs showing the change in surface phenotype of lymphocyte populations after 18t15-12s, 18t15-12s16 and 7t15-21s stimulation.

FIG. 164 is a set of graphs showing enhanced glycolysis of NK cells after 18t15-12s treatment.

FIG. 165 is a set of graphs showing elevated levels of phosphorylated STAT4 and phosphorylated STAT5 in NK cells after 18t15-12s stimulation.

FIG. 166 is a set of graphs showing that 18t15-12s overnight stimulation of NK cells resulted in enhanced cell metabolism.

FIGS. 167A-C are a set of graphs showing the immunostimulation of C57BL/6 mice after 2t2 treatment.

FIGS. 168A-B are a set of graphs showing the immunostimulation of C57BL/6 mice after TGFRt15-TGFRs treatment.

FIGS. 169A-C are a series of graphs showing ApoE fed Western diet and treated with TGFRt15-TGFRs or 2t2 ^-/- Tregs, NK cells and CD8 in mice ⁺ In vivo stimulation of T cells.

FIGS. 170A-B are a set of graphs showing that 2t2 induces spleen cell proliferation in C57BL/6 mice.

FIG. 171A-C is a set of graphs showing the immunostimulation of C57BL/6 mice after TGFRt15-TGFRs treatment.

FIGS. 172A-B are a set of graphs showing ApoE fed Western feed and treated with TGFRt15-TGFRs or 2t2 ^-/- NK cells and CD8 in mice ⁺ T cells are induced to proliferate in vivo.

FIG. 173 is a schematic and set of graphs showing that NK cells expanded by 7t15-21s and anti-antibodies persisted in NSG mice after treatment with 7t15-21, TGFRt15-TGFRs or 2t 2.

FIGS. 174A-B are a set of graphs showing the enhanced cytotoxicity of NK cells after treatment with TGFRt 15-TGFRs.

FIGS. 175A-B are a set of graphs showing that NK cells have enhanced ADCC activity after treatment of NK cells with TGFRt 15-TGFRs.

FIG. 176 is a graph of the in vitro killing of senescent B16F10 melanoma cells by TGFRt15-TGFRs/2t2 activated mouse NK cells.

FIGS. 177A-H are a set of graphs showing the anti-tumor activity of TGFRt15-TGFRs plus anti-TRP 1 antibody (TA 99) in combination with chemotherapy in a melanoma mouse model.

FIGS. 178A-C are a set of graphs showing 2t2 improvement of ApoE ^-/- Hyperglycemia induced by western diet in mice.

FIG. 179 is a set of graphs showing cell surface staining, which outlines NK cell differentiation into cytokine induced memory cells like NK cells (CIML-NK cells) after 18t15-12s stimulation and culture in rhIL-15.

FIG. 180 shows the upregulation of CD44 memory T cells. The upper panel shows the upregulation of CD44 memory T cells following treatment with TGFRt 15-TGFRs. The lower panel shows the up-regulation of CD44 memory T cells after treatment with 2T 2.

Figures 181A and 181B show improvement in hair regrowth following depilation in mice treated with 2t2 or IL-2. FIG. 181A shows skin pigmentation of PBS, 2t2 or IL-2 treated mice on day 10 after dehairing. FIG. 181B shows the percent pigmentation of mice treated with PBS, 2t2, or IL-2, as analyzed using imageJ software.

FIG. 182 shows skin pigmentation on day 14 after dehairing in PBS, 2t2 or IL-2 treated mice.

FIG. 183 shows a graph of Factor X (FX) activation after treatment with single-or multi-chain chimeric polypeptides.

Figure 184 shows the clotting time in Prothrombin Time (PT) tests for buffers containing different concentrations of inovin.

FIG. 185 shows clotting time of the multi-chain chimeric polypeptides in PT assays.

FIG. 186 shows clotting time of the multi-chain chimeric polypeptide when mixed with 32DB cells in PT assays.

FIG. 187 shows clotting time of the multi-chain chimeric polypeptides when mixed with human PBMC in a PT assay.

FIG. 188 shows the binding of 7t15-21s137L (long version) and 7t15-21s137L (short version) to CD137 (4.1 BB).

FIGS. 189A-189D show the detection of IL7, IL21, IL15 and 4.1BBL in 7t15-21s137L (long panel) by respective antibodies using ELISA.

FIG. 190 shows IL-15 activity of 7t15-21s137L (long panel) and 7t15-21s137L (short panel) as assessed by proliferation assay of CTLL2 cells containing IL2Rαβγ.

FIGS. 191A-191C show CD4 in response to 2t2 or IL2 treatment ⁺ CD25 ^hi T _reg Cell, CD4 ⁺ CD25 ^- T _con Cells or CD8 ⁺ T _con Human blood lymphocytes in the cells pStat5a respond. FIG. 191A shows CD4 ⁺ CD25 ^hi T _reg pSTAT5 response in cells. FIG. C191B shows CD4 ⁺ CD25 ^- T _con pSTAT5 response in cells. FIG. 191C shows CD8 ⁺ T _con pSTAT5 response in cells.

FIGS. 192A-192E are a set of images showing that treatment with IL-2 based molecules (2 t 2) induced hair follicle formation in a mouse model after dehairing. Fig. 192A is an image of depilation of only control mice performed after shaving, fig. 192B is an image of mice administered low dose IL-2 (1 mg/kg) after depilation, and fig. 192C-192E show images of 2t2 mice administered 0.3mg/kg (fig. 192C), 1mg/kg (fig. 192D) and 3mg/kg (fig. 192E) after depilation. Black arrows represent anagen hair follicles which will then extend into the dermis and promote hair growth.

Figure 193 shows the total number of anagen hair follicles counted per 10 fields per treatment group.

FIG. 194 is a graph showing that NK cells (relative to unexposed NK cells) from two different donors have different percentages of DNA demethylation after amplification with 7t15-21s+ anti-Tissue Factor (TF) antibody (IgG 1) (50 nM).

FIG. 195 is a set of graphs showing immunophenotypes from peripheral blood analysis 4 days after single dose treatment with TGFRt 15-TGFRs.

Fig. 196 is a set of graphs showing immunophenotypes from peripheral blood analysis 4 days after single dose treatment with TGFRt 15-TGFRs.

Fig. 197 is a graph showing analysis of β -Gal staining by FACS seven days after the second administration of TGFRt 15-TGFRs.

FIG. 198 is a set of graphs showing the levels of senescence markers in liver tissue determined using qPCR 7 days after the second administration of TGFRt 15-TGFRs.

FIG. 199 is a set of graphs showing the levels of senescence markers in kidney tissue determined using qPCR 7 days after the second administration of TGFRt 15-TGFRs.

Graph 200 is a set of graphs showing the levels of senescence markers in skin tissue determined using qPCR 7 days after the second administration of TGFRt 15-TGFRs.

Fig. 201 is a set of graphs showing the levels of senescence markers in lung tissue determined using qPCR 7 days after the second administration of TGFRt 15-TGFRs.

FIG. 202 is a set of histological images showing beta-Gal staining on kidney tissue 7 days after second treatment with TGFRt 15-TGFRs.

FIGS. 203A-203C show chemotherapy induced p21 in C57BL/6 mice ^CIP1 p21 senescence-associated gene expression. Fig. 203A is an exemplary schematic showing an experimental treatment regimen. FIGS. 203B and 203C are diagrams showing p21, respectively ^CIP1 Map of p21 expression in lung (B) and liver (C) tissues.

FIG. 204 is a set of graphs showing immunophenotype and cell proliferation following treatment with IL-15-based agents on day 3 post-treatment.

FIGS. 205A-205C are graphs showing that TGFRt15-TGFRs treatment reduced senescence-associated gene expression in C57BL/6 mice. These figures show p21 in lung (A and B) tissue, respectively ^CIP1 Expression of p21 and CD26 and p21 in liver (C) tissue ^CIP1 Expression of p 21.

FIG. 206 is a diagram showing CD4 ⁺ 、CD8 ⁺ And a panel of Treg cell percentages and proliferation.

FIG. 207 is a graph showing NK, CD19 ⁺ And a set of graphs of percentage of monocytes and proliferation.

FIGS. 208A-208C are graphs showing markers of aging p21 in lung and liver tissue ^CIP1 Graph of evaluation of p21 and CD 26. FIGS. 208A and 208B show lung p21 ^CIP1 p21 (a) and lung CD26 (B) senescence markers. FIG. 208C shows liver p21 ^CIP1 p21 senescence markers.

FIG. 209 shows a schematic representation of an exemplary TGFRt15 x-TGFRs complex generated by the interaction between TGF-RII/IL-15 RαSu and TGF-RII/TF/IL-15 Mut proteins.

FIG. 210 shows a schematic representation of the interaction between exemplary TGF-beta RII/IL-15RαSu and TGF-beta RII/TF/IL-15Mut proteins.

FIG. 211A is a graph showing the binding activity of TGFRt15-TGFRs on TGF-. Beta.1 and LAP.

FIG. 211B is a graph showing the binding activity of TGFRII/Fc to TGF-. Beta.1 and LAP.

FIG. 211C is a graph showing the binding activity of TGFRt15-TGFRs on TGF-. Beta.1 and LAP.

Fig. 211D is a graph showing the binding activity of TGFRt15-TGFRs on TGF- β1 and LAP.

FIG. 211E is a graph showing the binding activity of TGFRt15-TGFRs, and 7t15-21s on CTLL-2 cells.

FIG. 212A is a graph of TGFRt15-TGFRs and TGFRt15 x-TGFRs TGF- β1 blocking activity.

FIG. 212B is a graph of IL-15 bioactivity of TGFRt15-TGFRs and TGFRt 15-TGFRs.

FIG. 212C is a graph showing that TGF- β1, TGF- β2 and TGF- β3 each similarly inhibited IL-4 induced CTLL-2 growth in the absence of TGFRt 15-TGFRs.

FIG. 212D is a graph showing that TGFRt15-TGFRs significantly reverse the inhibition of IL-4 induced CTLL-2 cell growth by TGF- β1 and TGF- β3.

FIG. 213A shows that after incubation at 4 ℃, 25 ℃ or 37 ℃ for 10 days, the IL-15 domain of TGFRt15-TGFRs is not significantly damaged.

FIG. 213B shows that after incubation at 4 ℃, 25 ℃ or 37 ℃ for 10 days, the TGF-RII domain of TGFRt15-TGFRs is not significantly damaged.

FIG. 213C is a graph showing TGF-. Beta.1 neutralization activity of TGFRt15-TGFRs after incubation in human serum at 4 ℃, 25 ℃ or 37 ℃ for 10 days.

FIG. 213D is a graph showing IL-15 activity of TGFRt15-TGFRs after incubation in human serum at 4 ℃, 25 ℃ or 37 ℃ for 10 days.

FIG. 214A is a graph showing cell-mediated cytotoxicity that can be measured in assays using NK cells and the indicated constructs.

FIG. 214B is a graph showing cell-mediated cytotoxicity in assays using PMBC and the constructs shown.

FIG. 214C is a graph showing intracellular granzyme B production in an assay using NK cells and the constructs shown.

FIG. 214D is a graph showing intracellular granzyme B production in an assay using PBMC and the constructs shown.

FIG. 214E is a graph showing interferon-gamma production in assays using NK cells and the indicated constructs.

FIG. 214F is a graph showing interferon-gamma production in assays using PMBC and the constructs shown.

FIG. 215 is a graph showing the pharmacokinetics (half-life, t _1/2 ) Is a diagram of (a).

FIG. 216 is a graph showing the toxicity of TGFRt15-TGFRs in C57BL/6 mice.

FIG. 217 is a graph showing the anti-tumor activity of TGFRt15-TGFRs in a C57BL/6 murine melanoma model.

FIG. 218 shows the activity of TGFRt15-TGFRs in nine week old C57BL6/j male mice, where the mice were administered 50. Mu.l of bleomycin (2.5 mg/kg, single dose) by the oropharyngeal route, followed by subcutaneous administration of TGFRt15-TGFRs (3 mg/kg) on day 17 post bleomycin treatment.

Graph 219 shows fasting blood glucose levels in db/db mice 4 days after treatment with TGFRt15-TGFRs or TGFRt 15-TGFRs.

Graphs 220A-220C show TGF beta 1-3 levels in db/db mice 4 days after treatment with TGFRt15-TGFRs or TGFRt 15-TGFRs: tgfβ1 (fig. 220A), tgfβ2 (fig. 220B), and tgfβ3 (fig. 220C).

Fig. 221A-E show lymphocyte subpopulations in db/db mice 4 days after treatment with TGFRt15-TGFRs or TGFRt 15-TGFRs: blood NK cells (FIG. 221A), blood Ki67 ⁺ NK cells (FIG. 221B), and blood granzyme B ⁺ (GzmB ⁺ ) (FIG. 221C), blood CD8 ⁺ (FIG. 221D) and blood CD8 ⁺ Ki67 ⁺ T cells (fig. 221E).

Figure 222A shows the interaction of TGFRt15 x-TGFRs or TGFRt15-TGFRs with latent tgfβ1 (SLC) or with CD39 (control).

Panel 222B shows the interaction of TGFRt 15. Times. -TGFRs and TGFRII-Fc with latent TGF-beta 1.

Fig. 223 is a graph showing coagulation time of minovin (inovin) in PT measurement.

FIG. 224 is a graph showing the clotting time of TGFRt15-TGFRs in the PT assay.

FIG. 225 is a graph showing gene expression of the senescence markers PAI-1, IL-1 alpha, IL6 and IL-1 beta in kidneys and comparing young and PBS or TGFRt15-TGFRs treated aged mice with short and long term follow-up.

FIG. 226 is a graph showing gene expression of senescence markers IL-1α and IL6 in liver.

FIG. 227 shows protein expression of the senescence marker PAI-1 in kidney.

FIG. 228 is a graph showing that IL15SA (positive control) or TGFRt 15. Times. -TGFRs+IL15SA mediates CD3 in blood ⁺ CD8 ⁺ 、CD3 ^- NK1.1 ⁺ And CD3 ⁺ CD45 ⁺ An increase in the percentage of immune cells, whereas treatment with TGFRt15 x-TGFRs has little or no effect on the percentage of these cell populations.

FIG. 229 is a graph showing that IL15SA (positive control) or TGFRt 15. Times. -TGFRs+IL15SA mediates CD3 in spleen ⁺ CD8 ⁺ 、CD3 ^- NK1.1 ⁺ And CD3 ⁺ CD45 ⁺ An increase in the percentage of immune cells, whereas treatment with TGFRt15 x-TGFRs has little or no effect on the percentage of these cell populations.

FIG. 230A shows gene expression of senescence marker p21 in kidney and liver tissue after treatment with test article.

Fig. 230B shows gene expression of senescence marker PAI1 in kidney and liver tissues after study treatment.

FIG. 230C shows gene expression of the senescence marker IL-1α in kidney and liver tissues after study treatment.

FIG. 230D shows gene expression of senescence marker IL-6 in kidney and liver tissues after study treatment.

FIG. 231A shows CD4 after treatment with the indicated agents ⁺ 、CD8 ⁺ And Treg cell percentage and proliferation. FIG. 231B shows NK and CD19 after treatment with the indicated agents ⁺ And percentage of monocytes and proliferation.

Figure 232A shows an assessment of gene expression of senescence marker p21 in mouse lung tissue following chemotherapy and treatment with the indicated agents.

Figure 232B shows an assessment of gene expression of the senescence marker CD26 in mouse lung tissue following chemotherapy and treatment with the indicated agents.

Figure 232C shows an assessment of gene expression of senescence marker p21 in mouse liver tissue following chemotherapy and treatment with the indicated agents.

FIGS. 233A-B are graphs showing that TGFRt15-TGFRs treatment enhances immune cell proliferation, expansion and activation in peripheral blood of B16F10 tumor bearing mice.

FIG. 234 is a graph showing that TGFRt15-TGFRs treatment reduces TGF-beta levels in plasma of B16F10 tumor bearing mice.

FIG. 235 is a graph showing that TGFRt15-TGFRs treatment reduces the levels of pro-inflammatory cytokines in plasma of B16F10 tumor bearing mice.

FIG. 236 shows that TGFRt15-TGFRs treatment enhances NK and CD8 expression in the spleen of B16F10 tumor bearing mice.

FIGS. 237A-B show that TGFRt15-TGFRs treatment enhances glycolytic activity of B16F10 tumor bearing mouse spleen cells.

Figures 238A-B show that TGFRt15-TGFRs treatment enhances mitochondrial respiration of spleen cells in B16F10 tumor bearing mice.

Figures 239A-B show that TGFRt15-TGFRs treatment enhanced NK and CD8 immune cell infiltration (TILs) into tumors of B16F10 tumor bearing mice.

FIG. 240 shows histopathological analysis of tumors after TGFRt15-TGFRs treatment, wherein tumors showed less mitotic and necrotic activity after TGFRt 15-TGFRs+TA 99 antibody treatment. Mitotic index is related to dividing cells and the presence of necrosis is a measure of more invasive character and poor prognosis.

FIG. 241 is a graph showing anti-PD-L1 antibodies in combination with TGFRt 15-TGFRs+TA 99 antibodies and chemotherapy in a B16F10 melanoma mouse model.

FIG. 242 is a graph showing that the anti-tumor efficacy of TGFRt15-TGFRs is dependent on NK and CD 8T cells in a B16F10 melanoma mouse model.

Fig. 243A-B are graphs showing gene expression of senescence markers p21, IL-1 alpha, and IL6 in liver and lung tissues of tumor-bearing mice after chemotherapy.

FIG. 244 is a graph showing induction of gene expression of senescence markers p21, IL6, H2AX, and NK cell ligands Rae1e and ULBP1 by docetaxel-treated B16F10 GFP cells.

Fig. 245 shows tumor-infiltrating lymphocytes every day after 4 days of treatment in tumor-bearing mice.

Fig. 246A-B show flow cytometry analysis of tumor cells, indicating that mice treated with immunotherapy showed a smaller number of GFP-positive senescent tumor cells after 4 days and 10 days of treatment (fig. 246A) as compared to PBS control group, and tumor cells plated in 24-well plates were assessed by fluorescence microscopy (fig. 246B).

FIG. 247 shows TGF beta levels in kidney of mice after induction of kidney injury with cisplatin and treatment with TGFRt 15-TGFRs.

FIGS. 248A-C show the toxicological effects of repeated subcutaneous administration of TGFRt15-TGFRs in C57BL/6 mice. The change in body weight was shown by SD21 (fig. 248A). Spleen weights (FIG. 248B) and blood count and differences (FIG. 248C) of mice are shown at SD21 after one dose of TGFRt15-TGFRs and after two doses of TGFRt 15-TGFRs.

FIG. 249 shows the plasma levels of TGF- β isoforms in mice treated in vivo with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15-TGFRs (3 mg/kg).

Graphs 250A-B show the rate of change of glycolytic capacity (ECAR) (graph 250A) and mitochondrial respiration capacity (OCR) (graph 250B) in mouse spleen cells after in vivo treatment with PBS, TGFRt15-TGFRs or IL15 SA.

Graphs 251A-B show the rate of change of glycolytic capacity (ECAR) (graph 251A) and mitochondrial respiratory capacity (OCR) (graph 251B) in mouse spleen cells after in vitro treatment with PBS, TGFRt15-TGFRs or TGFRt 15-TGFRs.

Figures 252A-E show changes in tumor growth and survival of B16F10 melanoma in C57BL/6 mice after in vitro treatment with PBS, TGFRt15-TGFRs or TGFRt 15-TGFRs. Tumor volume (figure 252A) and mouse survival (based on<4000mm ³ Is a tumor volume of (c) (fig. 252B). As shown in figure 252A, mice were treated intraperitoneally with anti-CD 8, anti-NK, or anti-CD 8 and anti-NK antibodies for 1 week to deplete immune cells prior to injection of B16F10 melanoma tumor cells. Tumor bearing mice were then treated with PBS or 20mg/kg TGFRt15-TGFRs on day 1 and day 4 after tumor cell inoculation. Animals were assessed for tumor volume (panel 252C) and mouse survival (panel 252D). B16F10 tumor-bearing mice were treated with PBS or 20mg/kg of TGFRt15-TGFRs on days 1 and 7 post tumor inoculation (FIG. 252E). On day 11 after tumor inoculation, tumors were harvested and tumor-infiltrating NK1.1 by flow cytometry ⁺ Cell and CD8 ⁺ T cells were quantified.

Figures 253A-B show the effect of treatment on fasting blood glucose (figure 253A) and insulin (figure 253B) levels in db/db mice receiving PBS (control) or TGFRt 15-TGFRs.

FIG. 254A shows fold change in gene expression levels in the pancreas of db/db mice receiving TGFRt15-TGFRs as compared to PBS control.

FIGS. 254B-D show mean fold changes in the expression levels of SASP, senescence and beta cell index genes, respectively, in db/db mice pancreas receiving TGFRt15-TGFRs, as compared to PBS control.

Panels 255A-B show pancreas from db/db mice treated with PBS (control) (FIG. 255A) or TGFRt15-TGFRs (FIG. 255B)Multispectral imaging of glandular tissue sections. Representative islets, insulin ⁺ The islet beta cell is OPAL-520, insulin ⁺ p21 ⁺ Beta cells were OPAL-570 (shown as white blood cells in gray scale), which were reduced in the TGRt15-TGFRs treated group (fig. 255B) compared to the PBS treated group (fig. 255A). FIGS. 255C and 255D show islet insulin in pancreatic tissue sections from db/db mice treated with PBS (control) or TGFRt15-TGFRs ⁺ (FIG. 255C) and insulin ⁺ p21 ⁺ (FIG. 255D) level of cells.

Figures 256A-C show the effect of treatment on the percent of db/db mouse blood immune cell subpopulations that received PBS (control) or TGFRt 15-TGFRs.

FIG. 257 shows the percentage of Ki67 positive immune cells induced in blood after subcutaneous treatment of cynomolgus monkeys with TGFRt15-TGFRs, compared to PBS (vehicle).

FIG. 258 shows extracellular acidification rate (ECAR), representing glycolytic function of splenocytes isolated from young (6 week old) and aged (72 week old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15-TGFRs (3 mg/kg).

Fig. 259 shows Oxygen Consumption Rate (OCR), representing mitochondrial respiration of splenocytes isolated from young (6 week old) and aged (72 week old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15-TGFRs (3 mg/kg).

Figure 260 shows the percentage of immune cell subsets in blood from young (6 week old) and aged (72 week old) mice 4 days after treatment in vivo with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15-TGFRs (3 mg/kg).

Figure 261 shows the percentage of immune cell subpopulations in spleens from young (6 week old) and old (72 week old) mice 4 days after treatment with PBS, TGFRt15-TGFRs or TGFRt15 x-TGFRs in vivo.

FIG. 262 shows the gene expression levels of IL 1-alpha, IL 1-beta, IL-6, p21 and PAI-1 in the liver of aged mice after one or two doses of TGFRt15-TGFRs treatment.

FIG. 263 shows liver tissue inflammation scores of aged mice after one or two doses of TGFRt15-TGFRs treatment.

FIG. 264 shows the expression levels of IL 1-alpha, IL 1-beta, IL-6, IL-8, TGF-beta, PAI-1, collagen and fibronectin proteins in the liver of aged mice after treatment with one or two doses of TGFRt 15-TGFRs.

FIG. 265 shows the levels of β -galactosidase in liver tissue of aged 4 days old mice after in vivo treatment with PBS or TGFRt 15-TGFRs.

FIG. 266 shows the survival curve of 72 week old C57BL/6 mice after subcutaneous treatment with PBS or a dose of TGFRt15-TGFRs (3 mg/kg).

FIG. 267 shows protein levels of SASP factor in liver of B16F10 tumor bearing mice following chemotherapy and TGFRt 15-TGFRs+TA 99 therapy.

FIGS. 268A-B show CD8 after chemotherapy and TGFRt 15-TGFRs+TA 99 treatment ⁺ Depletion of T cell (dpCD 8) and NK cell (dpNK) antibodies TIS B16F10-GFP cells in tumors in mice (FIG. 268A), NK and CD8 ⁺ Effect of T cell (fig. 268B) level.

Figures 269A-E show the anti-tumor activity and mechanism of action of TGFRt 15-tgfrs+ta 99 combined immune checkpoint inhibitors in B16F10 tumor bearing mice. Fig. 269A shows an exemplary schematic for treating B16F10 melanoma in a mouse model. Figure 269B shows the volume and change in volume over time at day 18 following tumor administration of docetaxel followed by combination treatment comprising TGFRt15-TGFRs + ta99+ anti-PD-L1 antibody, as compared to PBS or chemotherapy alone treatment. FIGS. 269C and 269D show treatment of tumor-infiltrating CD28 at day 18 ⁺ CD8 ⁺ T cells and spleen IFN gamma ⁺ CD8 ⁺ Effect of T cell percentage. FIG. 269E shows treatment of CD8 invasive to tumors on day 18 ⁺ And CD8 ⁺ CD44 ^hi Effect of NKG2D levels (MFI) of T cells.

Figures 270A-D show changes in tumor growth and survival of SW1990 human pancreatic tumors in C57BL/6scid mice after in vitro treatment with PBS, gemcitabine and nab-paclitaxel chemotherapy, TGFRt15-TGFRs, or TGFRt15-tgfrs+ chemotherapy. Fig. 270A shows an exemplary schematic of the treatment of SW1990 human pancreatic tumors in a xenograft mouse model. Figures 270B and 270C show the volume and volume change over time at day 38 after tumor administration including the combination treatment of TGFRt15-TGFRs + chemotherapy, respectively, as compared to PBS or chemotherapy alone treatment. Figure 270D shows the effect of treatment on survival of mice bearing SW1990 human pancreatic tumors.

Detailed Description

Provided herein are methods of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors. Also provided herein are methods of reducing naturally occurring and/or treatment-induced accumulation of senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors. Also provided herein are methods of reducing the levels of markers of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors. Also provided herein are methods of attenuating the activity of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more agents that result in attenuation of activation of a TGF- β receptor. Also provided herein are methods of reducing the level or activity of a SASP factor derived from naturally occurring and/or treatment-induced aging cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of a TGF-beta receptor.

Also provided herein are methods of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. Also provided herein are methods of reducing accumulation of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. Also provided herein are methods of reducing the level of a marker of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. Also provided herein are methods of reducing the activity of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. Also provided herein are methods of reducing the level and/or activity of one or more SASP factors derived from naturally occurring and/or therapeutically induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Provided herein are methods of treating an aging-related disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of one or more Natural Killer (NK) cell activators and/or a therapeutically effective amount of activated NK cells to a subject identified as having an aging-related disease or disorder. Also provided herein are methods of killing or reducing the number of senescent cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more NK cell activators and/or a therapeutically effective amount of activated NK cells. Also provided herein are methods of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators and/or a therapeutically effective amount of activated NK cells. Also provided herein are methods of aiding in the treatment of obesity in a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators and/or a therapeutically effective amount of activated NK cells.

Activation of NK cells

Some embodiments of any of the methods described herein can include administering to a subject (e.g., any of the exemplary subjects described herein) a therapeutically effective amount of activated NK cells (e.g., human activated NK cells). An activated NK cell is an NK cell (e.g., a human NK cell) that exhibits elevated levels of expression of two or more (e.g., three, four, five, or six) of CD25, CD69, MTOR-C1, SREBP1, IFN- γ, and granzyme (e.g., granzyme B), e.g., as compared to a resting NK cell (e.g., a human resting NK cell). For example, the expression level of two or more (e.g., three, four, five, or six) of activated NK cells, CD25, CD69, MTOR-C1, SREBP1, IFN- γ, and granzyme (e.g., granzyme B) is increased by at least 10% (e.g., by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 120%, by at least 140%, by at least 160%, by at least 180%, by at least 200%, by at least 220%, by at least 240%, by at least 260%, by at least 280%, or by at least 300%) as compared to resting NK cells (e.g., human activated NK cells).

In some embodiments, the expression level of two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of CD25, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, CD, KIR2DS1, KIR2DS2/3, KIR2DL4, KIR2DS5, KIR3DS1, NKG2C, CCR, CXCR3, L-selectin, CXCR1, CXCR2, CX3CR1, chemR23, CXCR4, CCR5, S1P5, c-Kit, mTORC1 is optionally also increased by at least 10% (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, or 29) of activated NK cells as compared to resting NK cells (e.g., human activated NK cells), at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, or at least 300%.

For example, as compared to resting NK cells (e.g., human resting NK cells), activating two or more of CD25, CD69, mTOR-C1, SREBP1, IFN-gamma and granzyme (e.g., granzyme B) of NK cells (e.g., human activated NK cells) (e.g., three, four, five, or six) expression levels from about 10% to about 500%, from about 10% to about 450%, from about 10% to about 400%, from about 10% to about 350%, from about 10% to about 300%, from about 10% to about 280%, from about 10% to about 260%, from about 10% to about 240%, from about 10% to about 220%, from about 10% to about 200%, from about 10% to about 180%, from about 10% to about 160%, from about 10% to about 140%, from about 10% to about 120%, from about 10% to about 100%, from about 10% to about 80%, from about 10% to about 60%, from about 10% to about 40%, from about 10% to about 20%, from about about 20% to about 500%, about 20% to about 450%, about 20% to about 400%, about 20% to about 350%, about 20% to about 300%, about 20% to about 280%, about 20% to about 260%, about 20% to about 240%, about 20% to about 220%, about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 500%, about 40% to about 450%, about 40% to about 400%, about 40% to about 180%, about 160%, about, about 40% to about 350%, about 40% to about 300%, about 40% to about 280%, about 40% to about 260%, about 40% to about 240%, about 40% to about 220%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 80%, about 40% to about 60%, about 60% to about 500%, about 60% to about 450%, about 60% to about 400%, about 60% to about 350%, about 60% to about 300%, about 60% to about 280%, about 60% to about 260%, about 60% to about 240%, about 60% to about 220% i, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 60%, about 450%, about about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 80%, about 80% to about 500%, about 80% to about 450%, about 80% to about 400%, about 80% to about 350%, about 80% to about 300%, about 80% to about 280% about 80% to about 260%, about 80% to about 240%, about 80% to about 220%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 500%, about 100% to about 450%, about 100% to about 400%, about 100% to about 350%, about 100% to about 300%, about 100% to about 280%, about 100% to about 260%, about, about 100% to about 240%, about 100% to about 220%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 500%, about 120% to about 450%, about 120% to about 400%, about 120% to about 350%, about 120% to about 300%, about 120% to about 280%, about 120% to about 260%, about 120% to about 240%, about 120% to about 220%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 500%, about 140% to about 450%, about 140% to about 400%, about 140% to about 350%, about 140% to about 300%, about 140% to about 280%, about about 140% to about 260%, about 140% to about 240%, about 140% to about 220%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 500%, about 160% to about 450%, about 160% to about 400%, about 160% to about 350%, about 160% to about 300%, about 160% to about 280%, about 160% to about 260%, about 160% to about 240%, about 160% to about 220%, about 160% to about 200%, about 160% to about 180%, about 180% to about 500%, about 180% to about 450%, about 180% to about 400%, about 180% to about 350%, about 180% to about 300%, about 180% to about 280%, about 180% to about 260%, about 180% to about 240%, about 180% to about 220%, about, from about 180% to about 200%, from about 200% to about 500%, from about 200% to about 450%, from about 200% to about 400%, from about 200% to about 350%, from about 200% to about 300%, from about 200% to about 280%, from about 200% to about 260%, from about 200% to about 240%, from about 200% to about 220%, from about 220% to about 500%, from about 220% to about 450%, from about 220% to about 400%, from about 220% to about 350%, from about 220% to about 300%, from about 220% to about 280%, from about 220% to about 260%, from about 220% to about 240%, from about 240% to about 500%, from about 240% to about 450%, from about 240% to about 400%, from about 240% to about 350%, from about 240% to about 300%, from about 220% to about 450%, from about 400%, from about 220% to about about 240% to about 280%, about 240% to about 260%, about 260% to about 500%, about 260% to about 450%, about 260% to about 400%, about 260% to about 350%, about 260% to about 300%, about 260% to about 280%, about 280% to about 500%, about 280% to about 450%, about 280% to about 400%, about 280% to about 350%, about 280% to about 300%, about 300% to about 500%, about 300% to about 450%, about 300% to about 400%, about 300% to about 350%, about 350% to about 500%, about 350% to about 450%, about 350% to about 400%, about 400% to about 500%, about 400% to about 450%, or about 400% to about 500%.

In some embodiments, the expression level of two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of CD25, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, CD, KIR2DS1, KIR2DS2/3, KIR2DL4, KIR2DS5, KIR3DS1, NKG2C, CCR, CXCR3, L-selectin, CXCR1, CXCR2, CX3CR1, chemR23, CXCR4, CCR5, S1P5, c-Kit, mTORC1 in an activated NK cell (e.g., human) may also be increased by about 10% (e.g., to about any of the ranges described herein) as compared to resting NK cells.

Non-limiting examples of assays that can be used to determine the expression levels of CD25, CD69, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, CD, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS5, KIR3DS1, NKG2C, CCR7, CXCR3, L-selectin, CXCR1, CX3CR1, chemR23, CXCR4, CCR5, S1P5, c-Kit, mTORC1, MYC, SREBP1, IFN-gamma, and granzymes (e.g., granzymes B) include, for example, immunoblotting, fluorescence-assisted cell sorting, enzyme-linked immunosorbent assays, and RT-PCR.

Non-limiting examples of commercial ELISA assays that can be used to determine CD25 expression levels are available from Diaclone, covalab biotechnology company (Covalab Biotechnology) and Caltag medical systems company (Caltag Medsystems). The protein and cDNA sequences of mature human CD25 are shown below.

Mature human CD25 protein (SEQ ID NO: 1)

Human CD25 cDNA (SEQ ID NO: 2)

Non-limiting examples of commercial ELISA assays that can be used to determine CD69 expression levels are obtained from rapoo (RayBiotech), novus biologicals (Novus Biologicals), and Aviscera bioscience (Aviscera Bioscience). The protein and cDNA sequences of mature human CD69 are shown below.

Mature human CD69 protein (SEQ ID NO: 3)

Human CD69 cDNA (SEQ ID NO: 4)

The protein and cDNA sequences of mature human CD59 are shown below.

Mature human CD59 protein (SEQ ID NO: 5)

Human CD59 cDNA (SEQ ID NO: 6)

The protein and cDNA sequences of mature human CD352 are shown below.

Mature human CD352 protein (SEQ ID NO: 7)

Human CD352 cDNA (SEQ ID NO: 8)

The protein and cDNA sequences of mature human NKp80 are shown below.

Mature human NKp80 protein (SEQ ID NO: 9)

Human NKp80 cDNA (SEQ ID NO: 10)

Mature human DNAM-1 protein and cDNA sequences are shown below.

Mature human DNAM-1 protein (SEQ ID NO: 11)

Human DNAM-1 cDNA (SEQ ID NO: 12)

Mature human 2B4 protein and cDNA sequences are shown below.

Mature human 2B4 protein (SEQ ID NO: 13)

Human 2B4 cDNA (SEQ ID NO: 14)

Mature human NKp30 protein and cDNA sequences are shown below.

Mature human NKp30 protein (SEQ ID NO: 15)

Human NKp30 cDNA (SEQ ID NO: 16)

Mature human NKp44 protein and cDNA sequences are shown below.

Mature human NKp44 protein (SEQ ID NO: 17)

Human NKp44 cDNA (SEQ ID NO: 18)

Mature human NKp46 protein and cDNA sequences are shown below.

Mature human NKp46 protein (SEQ ID NO: 19)

Human NKp46 cDNA (SEQ ID NO: 20)

Mature human NKG2D protein and cDNA sequences are shown below.

Mature human NKG2D protein (SEQ ID NO: 21)

Human NKG2D cDNA (SEQ ID NO: 22)

Mature human CD16a protein and cDNA sequences are shown below.

Mature human CD16a protein (SEQ ID NO: 23)

Human CD16a cDNA (SEQ ID NO: 24)

atggctgagggcacactctggcagattctgtgtgtgtcctcagatgctcagccacagacctttgagggagtaaagggggcagacccacccaccttgcctccaggctctttccttcctggtcctgttctatggtggggctcccttgccagacttcagactgagaagtcagatgaagtttcaagaaaaggaaattggtgggtgacagagatgggtggaggggctggggaaaggctgtttacttcctcctgtctagtcggtttggtccctttagggctccggatatctttggtgacttgtccactccagtgtggcatcatgtggcagctgctcctcccaactgctctgctacttctagtttcagctggcatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagggtgctcgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagagcctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcgacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaaggcaggaagtattttcatcataattctgacttctacattccaaaagccacactcaaagacagcggctcctacttctgcagggggctttttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggcagtgtcaaccatctcatcattctttccacctgggtaccaagtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacattcgaagctcaacaagagactggaaggaccataaatttaaatggagaaaggaccctcaagacaaatga

Mature human CD16b protein and cDNA sequences are shown below.

Mature human CD16b protein (SEQ ID NO: 25)

Human CD16b cDNA (SEQ ID NO: 26)

atgtggcagctgctcctcccaactgctctgctacttctagtttcagctggcatgcggactgaagatctcccaaaggctgtggtgttcctggagcctcaatggtacagcgtgcttgagaaggacagtgtgactctgaagtgccagggagcctactcccctgaggacaattccacacagtggtttcacaatgagaacctcatctcaagccaggcctcgagctacttcattgacgctgccacagtcaacgacagtggagagtacaggtgccagacaaacctctccaccctcagtgacccggtgcagctagaagtccatatcggctggctgttgctccaggcccctcggtgggtgttcaaggaggaagaccctattcacctgaggtgtcacagctggaagaacactgctctgcataaggtcacatatttacagaatggcaaagacaggaagtattttcatcataattctgacttccacattccaaaagccacactcaaagatagcggctcctacttctgcagggggcttgttgggagtaaaaatgtgtcttcagagactgtgaacatcaccatcactcaaggtttggcagtgtcaaccatctcatcattctctccacctgggtaccaagtctctttctgcttggtgatggtactcctttttgcagtggacacaggactatatttctctgtgaagacaaacatttga

Mature human KIR2DS1 protein and cDNA sequences are shown below.

Human KIR2DS1 protein (SEQ ID NO: 27)

Human KIR2DS1 cDNA (SEQ ID NO: 28)

atgtcgctcacggtcgtcagcatggcgtgtgttgggttcttcttgctgcagggggcctggccacatgagggagtccacagaaaaccttccctcctggcccacccaggtcgcctggtgaaatcagaagagacagtcatcctgcaatgttggtcagatgtcatgtttgaacacttccttctgcacagagaggggatgtttaacgacactttgcgcctcattggagaacaccatgatggggtctccaaggccaacttctccatcagtcgcatgaagcaagacctggcagggacctacagatgctacggttctgttactcactccccctatcagttgtcagctcccagtgaccctctggacatcgtgatcataggtctatatgagaaaccttctctctcagcccagccgggccccacggttctggcaggagagaatgtgaccttgtcctgcagctcccggagctcctatgacatgtaccatctatccagggaaggggaggcccatgaacgtaggctccctgcagggaccaaggtcaacggaacattccaggccaactttcctctgggccctgccacccatggagggacctacagatgcttcggctctttccgtgactctccatacgagtggtcaaagtcaagtgacccactgcttgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagctccgaaaccggtaaccccagacacctacatgttctgattgggacctcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcgctggtgctccgacaaaaaaaatgctgctgtaatggaccaagagcctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtgtcatacgcataa

Mature human KIR2DS2 protein and cDNA sequences are shown below.

Mature human KIR2DS2 protein (SEQ ID NO: 29)

Human KIR2DS2 cDNA (SEQ ID NO: 30)

atgtcgctcatggtcgtcagcatggcgtgtgttgggttcttcttgctgcagggggcctggccacatgagggagtccacagaaaaccttccctcctggcccacccaggtcccctggtgaaatcagaagagacagtcatcctgcaatgttggtcagatgtcaggtttgagcacttccttctgcacagagaggggaagtataaggacactttgcacctcattggagagcaccatgatggggtctccaaggccaacttctccatcggtcccatgatgcaagaccttgcagggacctacagatgctacggttctgttactcactccccctatcagttgtcagctcccagtgaccctctggacatcgtcatcacaggtctatatgagaaaccttctctctcagcccagccgggccccacggttttggcaggagagagcgtgaccttgtcctgcagctcccggagctcctatgacatgtaccatctatccagggagggggaggcccatgaacgtaggttctctgcagggcccaaggtcaacggaacattccaggccgactttcctctgggccctgccacccacggaggaacctacagatgcttcggctctttccgtgactctccctatgagtggtcaaactcgagtgacccactgcttgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagctccaaaaccggtaaccccagacacctgcatgttctgattgggacctcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgctgtaatggaccaagagcctgcagggaacagaacagtgaacagcgaggactctgatgaacaagaccctcaggaggtgacatacacacagttgaatcactgcgttttcacacagagaaaaatcactcgcccttctcagaggcccaagacacccccaacagatatcatcgtgtacacggaacttccaaatgctgagtccaga

Mature human KIR2DS3 protein and cDNA sequences are shown below.

Mature human KIR2DS3 protein (SEQ ID NO: 31)

Human KIR2DS3 cDNA (SEQ ID NO: 32)

atgtcgctcatggtcatcagcatggcatgtgttgggttcttctggctgcagggggcctggccacatgagggattccgcagaaaaccttccctcctggcccacccaggtcgcctggtgaaatcagaagagacagtcatcctgcaatgttggtcagatgtcatgtttgagcacttccttctgcacagagaggggacgtttaacgacactttgcgcctcattggagagcacattgatggggtctccaaggccaacttctccatcggtcgcatgaggcaagacctggcagggacctacagatgctacggttctgttcctcactccccctatcagttttcagctcccagtgaccctctggacatcgtgatcacaggtctatatgagaaaccttctctctcagcccagccgggccccacggttctggcaggagagagcgtgaccttgtcctgcagctcctggagctcctatgacatgtaccatctatccacggagggggaggcccatgaacgtaggttctctgcagggcccaaggtcaacggaacattccaggccgactttcctctgggccctgccacccaaggaggaacctacagatgcttcggctctttccatgactctccctacgagtggtcaaagtcaagtgacccactgcttgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagctccaaaaccggtaaccccagacacctacacgttctgattgggacctcagtggtcaaactccctttcaccatcctcctcttctttctccttcatcgctggtgctccgacaaaaaaaatgcatctgtaatggaccaagggcctgcggggaacagaacagtgaacagggaggattctgatgaacaggaccatcaggaggtgtcatacgcataa

Mature human KIR2DL4 protein and cDNA sequences are shown below.

Mature human KIR2DL4 protein (SEQ ID NO: 33)

Human KIR2DL4 cDNA (SEQ ID NO: 34)

atgtccccttcacatgttgtggtcaatgtgtcaactgcacgatccgggcccctcaccacatcctctgcaccggtcagtcgagccgagtcactgcgtcctggcagcagaagctgcaccatgtccatgtcacccacggtcatcatcctggcatgtcttgggttcttcttggaccagagtgtgtgggcacacgtgggtggtcaggacaagcccttctgctctgcctggcccagcgctgtggtgcctcaaggaggacacgtgactcttcggtgtcactatcgtcgtgggtttaacatcttcacgctgtacaagaaagatggggtccctgtccctgagctctacaacagaatattctggaacagtttcctcattagccctgtgaccccagcacacgcagggacctacagatgtcgaggttttcacccgcactcccccactgagtggtcggcacccagcaaccccctggtgatcatggtcacaggtctatatgagaaaccttcgcttacagcccggccgggccccacggttcgcgcaggagagaacgtgaccttgtcctgcagctcccagagctcctttgacatctaccatctatccagggagggggaagcccatgaacttaggctccctgcagtgcccagcatcaatggaacattccaggccgacttccctctgggtcctgccacccacggagagacctacagatgcttcggctctttccatggatctccctacgagtggtcagacccgagtgacccactgcctgtttctgtcacaggaaacccttctagtagttggccttcacccactgaaccaagcttcaaaactggtatcgccagacacctgcatgctgtgattaggtactcagtggccatcatcctctttaccatccttcccttctttctccttcatcgctggtgctccaaaaaaaaagatgctgctgtaatgaaccaagagcctgcgggacacagaacagtgaacagggaggactctgatgaacaagaccctcaggaggtgacatacgcacagttggatcactgcattttcacacagagaaaaatcactggcccttctcagaggagcaagagaccctcaacagataccagcgtgtgtatagaacttccaaatgctgagcccagagcgttgtctcctgcccatgagcaccacagtcaggccttgatgggatcttctagggagacaacagccctgtctcaaacccagcttgccagctctaatgtaccagcagctggaatctga

Mature human KIR2DS4 protein and cDNA sequences are shown below.

Mature human KIR2DS4 protein (SEQ ID NO: 35)

Human KIR2DL4 cDNA (SEQ ID NO: 36)

atgtcgctcatggtcatcatcatggcgtgtgttgggttcttcttgctgcagggggcctggccacaggagggagtccacagaaaaccttccttcctggccctcccaggtcacctggtgaaatcagaagagacagtcatcctgcaatgttggtcggatgtcatgtttgagcacttccttctgcacagagaggggaagtttaacaacactttgcacctcattggagagcaccatgatggggtttccaaggccaacttctccattggtcccatgatgcctgtccttgcaggaacctacagatgctacggttctgttcctcactccccctatcagttgtcagctcccagtgaccctctggacatggtgatcataggtctatatgagaaaccttctctctcagcccagccgggccccacggttcaggcaggagagaatgtgaccttgtcctgcagctccatctatccagggaaggggaggcccatgaacgtaggctccctgcagtgcgcagcatcaacggaacattccaggccgactttcctctgggccctgccacccacggagggacctacagatgcttcggctctttccgtgacgctccctacgagtggtcaaactcgagtgatccactgcttgtttccgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagctccaaaaccggtaaccccagacacctacatgttctgattgggacctcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcgctggtgctccgacaaaaaaaatgctgctgtaatggaccaagagcctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtgtcatacgcataa

Mature human KIR2DS5 protein and cDNA sequences are shown below.

Mature human KIR2DS5 (SEQ ID NO: 37)

Human KIR2DS5 cDNA (SEQ ID NO: 38)

atgtcgctcatggtcatcagcatggcgtgtgttgcgttcttcttgctgcagggggcctggccacatgagggattccgcagaaaaccttccctcctggcccacccaggtcccctggtgaaatcagaagagacagtcatcctgcaatgttggtcagatgtcatgtttgagcacttccttctgcacagagaggggacgtttaaccacactttgcgcctcattggagagcacattgatggggtctccaagggcaacttctccatcggtcgcatgacacaagacctggcagggacctacagatgctacggttctgttactcactccccctatcagttgtcagcgcccagtgaccctctggacatcgtgatcacaggtctatatgagaaaccttctctctcagcccagccgggccccacggttctggcaggagagagcgtgaccttgtcctgcagctcccggagctcctatgacatgtaccatctatccagggaaggggaggcccatgaacgtaggctccctgcagggcccaaggtcaacagaacattccaggccgactttcctctggaccctgccacccacggagggacctacagatgcttcggctctttccgtgactctccatacgagtggtcaaagtcaagtgacccactgcttgtttctgtcacaggaaactcttcaaatagttggccttcacccactgaaccaagctccgaaaccggtaaccccagacacctacacgttctgattgggacctcagtggtcaaactccctttcaccatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgcatctgtaatggaccaagggcctgcggggaacagaacagtgaacagggaggattctgatgaacaggaccatcaggaggtgtcatacgcataa

Mature human KIR3DS1 protein and cDNA sequences are shown below.

Mature human KIR3DS1 cDNA (SEQ ID NO: 39)

Human KIR3DS1 cDNA (SEQ ID NO: 40)

atgttgctcatggtcgtcagcatggcgtgtgttgggttgttcttggtccagagggccggtccacacatgggtggtcaggacaagcccttcctgtctgcctggcccagcgctgtggtgcctcgcggaggacacgtgactcttcggtgtcactatcgtcataggtttaacaatttcatgctatacaaagaagacagaatccacgttcccatcttccatggcagaatattccaggagggcttcaacatgagccctgtgaccacagcacatgcagggaactacacatgtcggggttcacacccacactcccccactgggtggtcggcacccagcaaccccatggtgatcatggtcacaggaaaccacagaaaaccttccctcctggcccacccaggtcccctggtgaaatcaggagagagagtcatcctgcaatgttggtcagatatcatgtttgagcacttctttctgcacaaagagtggatctctaaggacccctcacgcctcgttggacagatccatgatggggtctccaaggccaatttctccatcggttccatgatgcgtgcccttgcagggacctacagatgctacggttctgttactcacaccccctatcagttgtcagctcccagtgatcccctggacatcgtggtcacaggtctatatgagaaaccttctctctcagcccagccgggccccaaggttcaggcaggagagagcgtgaccttgtcctgtagctcccggagctcctatgacatgtaccatctatccagggaggggggagcccatgaacgtaggctccctgcagtgcgcaaggtcaacagaacattccaggcagatttccctctgggccctgccacccacggagggacctacagatgcttcggctctttccgtcactctccctacgagtggtcagacccgagtgacccactgcttgtttctgtcacaggaaacccttcaagtagttggccttcacccacagaaccaagctccaaatctggtaacctcagacacctgcacattctgattgggacctcagtggtcaaaatccctttcaccatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaaatgctgctgtaatggaccaagagcctgcagggaacagaagtga

Mature human NKG2C protein and cDNA sequences are shown below.

Mature human NKG2C protein (SEQ ID NO: 41)

Human NKG2C cDNA (SEQ ID NO: 42)

The mature human CCR7 protein and cDNA sequences of atgaataaacaaagaggaaccttctcagaagtgagtctggcccaggacccaaagcggcagcaaaggaaacctaaaggcaataaaagctccatttcaggaaccgaacaggaaatattccaagtagaattaaatcttcaaaatccttccctgaatcatcaagggattgataaaatatatgactgccaaggtttactgccacctccagagaagctcactgccgaggtcctaggaatcatttgcattgtcctgatggccactgtgttaaaaacaatagttcttattcctttcctggagcagaacaatttttccccgaatacaagaacgcagaaagcacgtcattgtggccattgtcctgaggagtggattacatattccaacagttgttattacattggtaaggaaagaagaacttgggaagagagtttgctggcctgtacttcgaagaactccagtctgctttctatagataatgaagaagaaatgaaatttctggccagcattttaccttcctcatggattggtgtgtttcgtaacagcagtcatcatccatgggtgacaataaatggtttggctttcaaacataagataaaagactcagataatgctgaacttaactgtgcagtgctacaagtaaatcgacttaaatcagcccagtgtggatcttcaatgatatatcattgtaagcataagctttag are shown below.

Mature human CCR7 protein (SEQ ID NO: 43)

Human CCR7 cDNA (SEQ ID NO: 44)

atggacctggggaaaccaatgaaaagcgtgctggtggtggctctccttgtcattttccaggtatgcctgtgtcaagatgaggtcacggacgattacatcggagacaacaccacagtggactacactttgttcgagtctttgtgctccaagaaggacgtgcggaactttaaagcctggttcctccctatcatgtactccatcatttgtttcgtgggcctactgggcaatgggctggtcgtgttgacctatatctatttcaagaggctcaagaccatgaccgatacctacctgctcaacctggcggtggcagacatcctcttcctcctgacccttcccttctgggcctacagcgcggccaagtcctgggtcttcggtgtccacttttgcaagctcatctttgccatctacaagatgagcttcttcagtggcatgctcctacttctttgcatcagcattgaccgctacgtggccatcgtccaggctgtctcagctcaccgccaccgtgcccgcgtccttctcatcagcaagctgtcctgtgtgggcatctggatactagccacagtgctctccatcccagagctcctgtacagtgacctccagaggagcagcagtgagcaagcgatgcgatgctctctcatcacagagcatgtggaggcctttatcaccatccaggtggcccagatggtgatcggctttctggtccccctgctggccatgagcttctgttaccttgtcatcatccgcaccctgctccaggcacgcaactttgagcgcaacaaggccatcaaggtgatcatcgctgtggtcgtggtcttcatagtcttccagctgccctacaatggggtggtcctggcccagacggtggccaacttcaacatcaccagtagcacctgtgagctcagtaagcaactcaacatcgcctacgacgtcacctacagcctggcctgcgtccgctgctgcgtcaaccctttcttgtacgccttcatcggcgtcaagttccgcaacgatctcttcaagctcttcaaggacctgggctgcctcagccaggagcagctccggcagtggtcttcctgtcggcacatccggcgctcctccatgagtgtggaggccgagaccaccaccaccttctccccatag

Mature human CXCR3 protein and cDNA sequences are shown below.

Mature human CXCR3 protein (SEQ ID NO: 45)

Human CXCR3 cDNA (SEQ ID NO: 46)

atggagttgaggaagtacggccctggaagactggcggggacagttataggaggagctgctcagagtaaatcacagactaaatcagactcaatcacaaaagagttcctgccaggcctttacacagccccttcctccccgttcccgccctcacaggtgagtgaccaccaagtgctaaatgacgccgaggttgccgccctcctggagaacttcagctcttcctatgactatggagaaaacgagagtgactcgtgctgtacctccccgccctgcccacaggacttcagcctgaacttcgaccgggccttcctgccagccctctacagcctcctctttctgctggggctgctgggcaacggcgcggtggcagccgtgctgctgagccggcggacagccctgagcagcaccgacaccttcctgctccacctagctgtagcagacacgctgctggtgctgacactgccgctctgggcagtggacgctgccgtccagtgggtctttggctctggcctctgcaaagtggcaggtgccctcttcaacatcaacttctacgcaggagccctcctgctggcctgcatcagctttgaccgctacctgaacatagttcatgccacccagctctaccgccgggggcccccggcccgcgtgaccctcacctgcctggctgtctgggggctctgcctgcttttcgccctcccagacttcatcttcctgtcggcccaccacgacgagcgcctcaacgccacccactgccaatacaacttcccacaggtgggccgcacggctctgcgggtgctgcagctggtggctggctttctgctgcccctgctggtcatggcctactgctatgcccacatcctggccgtgctgctggtttccaggggccagcggcgcctgcgggccatgcggctggtggtggtggtcgtggtggcctttgccctctgctggaccccctatcacctggtggtgctggtggacatcctcatggacctgggcgctttggcccgcaactgtggccgagaaagcagggtagacgtggccaagtcggtcacctcaggcctgggctacatgcactgctgcctcaacccgctgctctatgcctttgtaggggtcaagttccgggagcggatgtggatgctgctcttgcgcctgggctgccccaaccagagagggctccagaggcagccatcgtcttcccgccgggattcatcctggtctgagacctcagaggcctcctactcgggcttgtga

Mature human L-selectin protein and cDNA sequences are shown below.

Mature human L-selectin protein (SEQ ID NO: 47)

Human L-selectin cDNA (SEQ ID NO: 48)

atgggctgcagaagaactagagaaggaccaagcaaagccatgatatttccatggaaatgtcagagcacccagagggacttatggaacatcttcaagttgtgggggtggacaatgctctgttgtgatttcctggcacatcatggaaccgactgctggacttaccattattctgaaaaacccatgaactggcaaagggctagaagattctgccgagacaattacacagatttagttgccatacaaaacaaggcggaaattgagtatctggagaagactctgcctttcagtcgttcttactactggataggaatccggaagataggaggaatatggacgtgggtgggaaccaacaaatctcttactgaagaagcagagaactggggagatggtgagcccaacaacaagaagaacaaggaggactgcgtggagatctatatcaagagaaacaaagatgcaggcaaatggaacgatgacgcctgccacaaactaaaggcagccctctgttacacagcttcttgccagccctggtcatgcagtggccatggagaatgtgtagaaatcatcaataattacacctgcaactgtgatgtggggtactatgggccccagtgtcagtttgtgattcagtgtgagcctttggaggccccagagctgggtaccatggactgtactcaccctttgggaaacttcagcttcagctcacagtgtgccttcagctgctctgaaggaacaaacttaactgggattgaagaaaccacctgtggaccatttggaaactggtcatctccagaaccaacctgtcaagtgattcagtgtgagcctctatcagcaccagatttggggatcatgaactgtagccatcccctggccagcttcagctttacctctgcatgtaccttcatctgctcagaaggaactgagttaattgggaagaagaaaaccatttgtgaatcatctggaatctggtcaaatcctagtccaatatgtcaaaaattggacaaaagtttctcaatgattaaggagggtgattataaccccctcttcattccagtggcagtcatggttactgcattctctgggttggcatttatcatttggctggcaaggagattaaaaaaaggcaagaaatccaagagaagtatgaatgacccatattaa

Mature human CXCR1 protein and cDNA sequences are shown below.

Mature human CXCR1 protein (SEQ ID NO: 49)

Human CXCR1 cDNA (SEQ ID NO: 50)

atgtcaaatattacagatccacagatgtgggattttgatgatctaaatttcactggcatgccacctgcagatgaagattacagcccctgtatgctagaaactgagacactcaacaagtatgttgtgatcatcgcctatgccctagtgttcctgctgagcctgctgggaaactccctggtgatgctggtcatcttatacagcagggtcggccgctccgtcactgatgtctacctgctgaacctggccttggccgacctactctttgccctgaccttgcccatctgggccgcctccaaggtgaatggctggatttttggcacattcctgtgcaaggtggtctcactcctgaaggaagtcaacttctacagtggcatcctgctgttggcctgcatcagtgtggaccgttacctggccattgtccatgccacacgcacactgacccagaagcgtcacttggtcaagtttgtttgtcttggctgctggggactgtctatgaatctgtccctgcccttcttccttttccgccaggcttaccatccaaacaattccagtccagtttgctatgaggtcctgggaaatgacacagcaaaatggcggatggtgttgcggatcctgcctcacacctttggcttcatcgtgccgctgtttgtcatgctgttctgctatggattcaccctgcgtacactgtttaaggcccacatggggcagaagcaccgagccatgagggtcatctttgctgtcgtcctcatcttcctgctttgctggctgccctacaacctggtcctgctggcagacaccctcatgaggacccaggtgatccaggagagctgtgagcgccgcaacaacatcggccgggccctggatgccactgagattctgggatttctccatagctgcctcaaccccatcatctacgccttcatcggccaaaattttcgccatggattcctcaagatcctggctatgcatggcctggtcagcaaggagttcttggcacgtcatcgtgttacctcctacacttcttcgtctgtcaatgtctcttccaacctctga

Mature human CXCR2 protein and cDNA sequences are shown below.

Mature human CXCR2 protein (SEQ ID NO: 51)

Human CXCR2 cDNA (SEQ ID NO: 52) atggaagattttaacatggagagtgacagctttgaagatttctggaaaggtgaagatcttagtaattacagttacagctctaccctgcccccttttctactagatgccgccccatgtgaaccagaatccctggaaatcaacaagtattttgtggtcattatctatgccctggtattcctgctgagcctgctgggaaactccctcgtgatgctggtcatcttatacagcagggtcggccgctccgtcactgatgtctacctgctgaacctagccttggccgacctactctttgccctgaccttgcccatctgggccgcctccaaggtgaatggctggatttttggcacattcctgtgcaaggtggtctcactcctgaaggaagtcaacttctatagtggcatcctgctactggcctgcatcagtgtggaccgttacctggccattgtccatgccacacgcacactgacccagaagcgctacttggtcaaattcatatgtctcagcatctggggtctgtccttgctcctggccctgcctgtcttacttttccgaaggaccgtctactcatccaatgttagcccagcctgctatgaggacatgggcaacaatacagcaaactggcggatgctgttacggatcctgccccagtcctttggcttcatcgtgccactgctgatcatgctgttctgctacggattcaccctgcgtacgctgtttaaggcccacatggggcagaagcaccgggccatgcgggtcatctttgctgtcgtcctcatcttcctgctctgctggctgccctacaacctggtcctgctggcagacaccctcatgaggacccaggtgatccaggagacctgtgagcgccgcaatcacatcgaccgggctctggatgccaccgagattctgggcatccttcacagctgcctcaaccccctcatctacgccttcattggccagaagtttcgccatggactcctcaagattctagctatacatggcttgatcagcaaggactccctgcccaaagacagcaggccttcctttgttggctcttcttcagggcacacttccactactctctaa

Mature human CX3CR1 protein and cDNA sequences are shown below.

Mature human CX3CR1 protein (SEQ ID NO: 53)

Human CX3CR1 cDNA (SEQ ID NO: 54)

atggatcagttccctgaatcagtgacagaaaactttgagtacgatgatttggctgaggcctgttatattggggacatcgtggtctttgggactgtgttcctgtccatattctactccgtcatctttgccattggcctggtgggaaatttgttggtagtgtttgccctcaccaacagcaagaagcccaagagtgtcaccgacatttacctcctgaacctggccttgtctgatctgctgtttgtagccactttgcccttctggactcactatttgataaatgaaaagggcctccacaatgccatgtgcaaattcactaccgccttcttcttcatcggcttttttggaagcatattcttcatcaccgtcatcagcattgataggtacctggccatcgtcctggccgccaactccatgaacaaccggaccgtgcagcatggcgtcaccatcagcctaggcgtctgggcagcagccattttggtggcagcaccccagttcatgttcacaaagcagaaagaaaatgaatgccttggtgactaccccgaggtcctccaggaaatctggcccgtgctccgcaatgtggaaacaaattttcttggcttcctactccccctgctcattatgagttattgctacttcagaatcatccagacgctgttttcctgcaagaaccacaagaaagccaaagccattaaactgatccttctggtggtcatcgtgtttttcctcttctggacaccctacaacgttatgattttcctggagacgcttaagctctatgacttctttcccagttgtgacatgaggaaggatctgaggctggccctcagtgtgactgagacggttgcatttagccattgttgcctgaatcctctcatctatgcatttgctggggagaagttcagaagatacctttaccacctgtatgggaaatgcctggctgtcctgtgtgggcgctcagtccacgttgatttctcctcatctgaatcacaaaggagcaggcatggaagtgttctgagcagcaattttacttaccacacgagtgatggagatgcattgctccttctctga

Mature human ChemR23 protein and cDNA sequences are shown below.

Mature human ChemR23 protein (SEQ ID NO: 55)

Human ChemR23 cDNA (SEQ ID NO: 56)

atgagaatggaggatgaagattacaacacttccatcagttacggtgatgaataccctgattatttagactccattgtggttttggaggacttatcccccttggaagccagggtgaccaggatcttcctggtggtggtctacagcatcgtctgcttcctcgggattctgggcaatggtctggtgatcatcattgccaccttcaagatgaagaagacagtgaacatggtctggttcctcaacctggcagtggcagatttcctgttcaacgtcttcctcccaatccatatcacctatgccgccatggactaccactgggttttcgggacagccatgtgcaagatcagcaacttccttctcatccacaacatgttcaccagcgtcttcctgctgaccatcatcagctctgaccgctgcatctctgtgctcctccctgtctggtcccagaaccaccgcagcgttcgcctggcttacatggcctgcatggtcatctgggtcctggctttcttcttgagttccccatctctcgtcttccgggacacagccaacctgcatgggaaaatatcctgcttcaacaacttcagcctgtccacacctgggtcttcctcgtggcccactcactcccaaatggaccctgtggggtatagccggcacatggtggtgactgtcacccgcttcctctgtggcttcctggtcccagtcctcatcatcacagcttgctacctcaccatcgtgtgcaaactgcagcgcaaccgcctggccaagaccaagaagcccttcaagattattgtgaccatcatcattaccttcttcctctgctggtgcccctaccacacactcaacctcctagagctccaccacactgccatgcctggctctgtcttcagcctgggtttgcccctggccactgcccttgccattgccaacagctgcatgaaccccattctgtatgttttcatgggtcaggacttcaagaagttcaaggtggccctcttctctcgcctggtcaatgctctaagtgaagatacaggccactcttcctaccccagccatagaagctttaccaagatgtcatcaatgaatgagaggacttctatgaatgagagggagaccggcatgctttga

The protein and cDNA sequences of mature human CXCR4 are shown below.

Mature human CXCR4 protein (SEQ ID NO: 57)

Human CXCR4 cDNA (SEQ ID NO: 58)

atgtccattcctttgcctcttttgcagatatacacttcagataactacaccgaggaaatgggctcaggggactatgactccatgaaggaaccctgtttccgtgaagaaaatgctaatttcaataaaatcttcctgcccaccatctactccatcatcttcttaactggcattgtgggcaatggattggtcatcctggtcatgggttaccagaagaaactgagaagcatgacggacaagtacaggctgcacctgtcagtggccgacctcctctttgtcatcacgcttcccttctgggcagttgatgccgtggcaaactggtactttgggaacttcctatgcaaggcagtccatgtcatctacacagtcaacctctacagcagtgtcctcatcctggccttcatcagtctggaccgctacctggccatcgtccacgccaccaacagtcagaggccaaggaagctgttggctgaaaaggtggtctatgttggcgtctggatccctgccctcctgctgactattcccgacttcatctttgccaacgtcagtgaggcagatgacagatatatctgtgaccgcttctaccccaatgacttgtgggtggttgtgttccagtttcagcacatcatggttggccttatcctgcctggtattgtcatcctgtcctgctattgcattatcatctccaagctgtcacactccaagggccaccagaagcgcaaggccctcaagaccacagtcatcctcatcctggctttcttcgcctgttggctgccttactacattgggatcagcatcgactccttcatcctcctggaaatcatcaagcaagggtgtgagtttgagaacactgtgcacaagtggatttccatcaccgaggccctagctttcttccactgttgtctgaaccccatcctctatgctttccttggagccaaatttaaaacctctgcccagcacgcactcacctctgtgagcagagggtccagcctcaagatcctctccaaaggaaagcgaggtggacattcatctgtttccactgagtctgagtcttcaagttttcactccagctaa

Mature human CCR5 protein and cDNA sequences are shown below.

Mature human CCR5 protein (SEQ ID NO: 59)

Human CCR5 cDNA (SEQ ID NO: 60)

atggattatcaagtgtcaagtccaatctatgacatcaattattatacatcggagccctgccaaaaaatcaatgtgaagcaaatcgcagcccgcctcctgcctccgctctactcactggtgttcatctttggttttgtgggcaacatgctggtcatcctcatcctgataaactgcaaaaggctgaagagcatgactgacatctacctgctcaacctggccatctctgacctgtttttccttcttactgtccccttctgggctcactatgctgccgcccagtgggactttggaaatacaatgtgtcaactcttgacagggctctattttataggcttcttctctggaatcttcttcatcatcctcctgacaatcgataggtacctggctgtcgtccatgctgtgtttgctttaaaagccaggacggtcacctttggggtggtgacaagtgtgatcacttgggtggtggctgtgtttgcgtctctcccaggaatcatctttaccagatctcaaaaagaaggtcttcattacacctgcagctctcattttccatacagtcagtatcaattctggaagaatttccagacattaaagatagtcatcttggggctggtcctgccgctgcttgtcatggtcatctgctactcgggaatcctaaaaactctgcttcggtgtcgaaatgagaagaagaggcacagggctgtgaggcttatcttcaccatcatgattgtttattttctcttctgggctccctacaacattgtccttctcctgaacaccttccaggaattctttggcctgaataattgcagtagctctaacaggttggaccaagctatgcaggtgacagagactcttgggatgacgcactgctgcatcaaccccatcatctatgcctttgtcggggagaagttcagaaactacctcttagtcttcttccaaaagcacattgccaaacgcttctgcaaatgctgttctattttccagcaagaggctcccgagcgagcaagctcagtttacacccgatccactggggagcaggaaatatctgtgggcttgtga

Mature human S1P5 protein and cDNA sequences are shown below.

Mature human S1P5 protein (SEQ ID NO: 61)

Human S1P5 cDNA (SEQ ID NO: 62)

atggagtcggggctgctgcggccggcgccggtgagcgaggtcatcgtcctgcattacaactacaccggcaagctccgcggtgcgcgctaccagccgggtgccggcctgcgcgccgacgccgtggtgtgcctggcggtgtgcgccttcatcgtgctagagaatctagccgtgttgttggtgctcggacgccacccgcgcttccacgctcccatgttcctgctcctgggcagcctcacgttgtcggatctgctggcaggcgccgcctacgccgccaacatcctactgtcggggccgctcacgctgaaactgtcccccgcgctctggttcgcacgggagggaggcgtcttcgtggcactcactgcgtccgtgctgagcctcctggccatcgcgctggagcgcagcctcaccatggcgcgcagggggcccgcgcccgtctccagtcgggggcgcacgctggcgatggcagccgcggcctggggcgtgtcgctgctcctcgggctcctgccagcgctgggctggaattgcctgggtcgcctggacgcttgctccactgtcttgccgctctacgccaaggcctacgtgctcttctgcgtgctcgccttcgtgggcatcctggccgctatctgtgcactctacgcgcgcatctactgccaggtacgcgccaacgcgcggcgcctgccggcacggcccgggactgcggggaccacctcgacccgggcgcgtcgcaagccgcgctcgctggccttgctgcgcacgctcagcgtggtgctcctggcctttgtggcatgttggggccccctcttcctgctgctgttgctcgacgtggcgtgcccggcgcgcacctgtcctgtactcctgcaggccgatcccttcctgggactggccatggccaactcacttctgaaccccatcatctacacgctcaccaaccgcgacctgcgccacgcgctcctgcgcctggtctgctgcggacgccactcctgcggcagagacccgagtggctcccagcagtcggcgagcgcggctgaggcttccgggggcctgcgccgctgcctgcccccgggccttgatgggagcttcagcggctcggagcgctcatcgccccagcgcgacgggctggacaccagcggctccacaggcagccccggtgcacccacagccgcccggactctggtatcagaaccggctgcagactga

Mature human C-kit protein and cDNA sequences are shown below.

Mature human C-kit protein (SEQ ID NO: 63)

Human C-kit cDNA (SEQ ID NO: 64)

atgagaggcgctcgcggcgcctgggattttctctgcgttctgctcctactgcttcgcgtccagacaggctcttctcaaccatctgtgagtccaggggaaccgtctccaccatccatccatccaggaaaatcagacttaatagtccgcgtgggcgacgagattaggctgttatgcactgatccgggctttgtcaaatggacttttgagatcctggatgaaacgaatgagaataagcagaatgaatggatcacggaaaaggcagaagccaccaacaccggcaaatacacgtgcaccaacaaacacggcttaagcaattccatttatgtgtttgttagagatcctgccaagcttttccttgttgaccgctccttgtatgggaaagaagacaacgacacgctggtccgctgtcctctcacagacccagaagtgaccaattattccctcaaggggtgccaggggaagcctcttcccaaggacttgaggtttattcctgaccccaaggcgggcatcatgatcaaaagtgtgaaacgcgcctaccatcggctctgtctgcattgttctgtggaccaggagggcaagtcagtgctgtcggaaaaattcatcctgaaagtgaggccagccttcaaagctgtgcctgttgtgtctgtgtccaaagcaagctatcttcttagggaaggggaagaattcacagtgacgtgcacaataaaagatgtgtctagttctgtgtactcaacgtggaaaagagaaaacagtcagactaaactacaggagaaatataatagctggcatcacggtgacttcaattatgaacgtcaggcaacgttgactatcagttcagcgagagttaatgattctggagtgttcatgtgttatgccaataatacttttggatcagcaaatgtcacaacaaccttggaagtagtagataaaggattcattaatatcttccccatgataaacactacagtatttgtaaacgatggagaaaatgtagatttgattgttgaatatgaagcattccccaaacctgaacaccagcagtggatctatatgaacagaaccttcactgataaatgggaagattatcccaagtctgagaatgaaagtaatatcagatacgtaagtgaacttcatctaacgagattaaaaggcaccgaaggaggcacttacacattcctagtgtccaattctgacgtcaatgctgccatagcatttaatgtttatgtgaatacaaaaccagaaatcctgacttacgacaggctcgtgaatggcatgctccaatgtgtggcagcaggattcccagagcccacaatagattggtatttttgtccaggaactgagcagagatgctctgcttctgtactgccagtggatgtgcagacactaaactcatctgggccaccgtttggaaagctagtggttcagagttctatagattctagtgcattcaagcacaatggcacggttgaatgtaaggcttacaacgatgtgggcaagacttctgcctattttaactttgcatttaaaggtaacaacaaagagcaaatccatccccacaccctgttcactcctttgctgattggtttcgtaatcgtagctggcatgatgtgcattattgtgatgattctgacctacaaatatttacagaaacccatgtatgaagtacagtggaaggttgttgaggagataaatggaaacaattatgtttacatagacccaacacaacttccttatgatcacaaatgggagtttcccagaaacaggctgagttttgggaaaaccctgggtgctggagctttcgggaaggttgttgaggcaactgcttatggcttaattaagtcagatgcggccatgactgtcgctgtaaagatgctcaagccgagtgcccatttgacagaacgggaagccctcatgtctgaactcaaagtcctgagttaccttggtaatcacatgaatattgtgaatctacttggagcctgcaccattggagggcccaccctggtcattacagaatattgttgctatggtgatcttttgaattttttgagaagaaaacgtgattcatttatttgttcaaagcaggaagatcatgcagaagctgcactttataagaatcttctgcattcaaaggagtcttcctgcagcgatagtactaatgagtacatggacatgaaacctggagtttcttatgttgtcccaaccaaggccgacaaaaggagatctgtgagaataggctcatacatagaaagagatgtgactcccgccatcatggaggatgacgagttggccctagacttagaagacttgctgagcttttcttaccaggtggcaaagggcatggctttcctcgcctccaagaattgtattcacagagacttggcagccagaaatatcctccttactcatggtcggatcacaaagatttgtgattttggtctagccagagacatcaagaatgattctaattatgtggttaaaggaaacgctcgactacctgtgaagtggatggcacctgaaagcattttcaactgtgtatacacgtttgaaagtgacgtctggtcctatgggatttttctttgggagctgttctctttaggaagcagcccctatcctggaatgccggtcgattctaagttctacaagatgatcaaggaaggcttccggatgctcagccctgaacacgcacctgctgaaatgtatgacataatgaagacttgctgggatgcagatcccctaaaaagaccaacattcaagcaaattgttcagctaattgagaagcagatttcagagagcaccaatcatatttactccaacttagcaaactgcagccccaaccgacagaagcccgtggtagaccattctgtgcggatcaattctgtcggcagcaccgcttcctcctcccagcctctgcttgtgcacgacgatgtctga

The protein and cDNA sequences of mature human mTOR are shown below.

Mature human mTOR protein (SEQ ID NO: 65)

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Human mTOR cDNA (SEQ ID NO: 66)

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Non-limiting examples of commercial ELISA assays that can be used to determine SREBP1 expression levels are obtained from Novus biologicals and Ai Bokang (Abcam). Mature human SREBP1 protein and cDNA sequences are shown below.

Mature human SREBP1 protein (SEQ ID NO: 67)

MDEPPFSEAALEQALGEPCDLDAALLTDIEDMLQLINNQDSDFPGLFDPPYAGSGAGGTDPASPDTSSPGSLSPPPATLSSSLEAFLSGPQAAPSPLSPPQPAPTPLKMYPSMPAFSPGPGIKEESVPLSILQTPTPQPLPGALLPQSFPAPAPPQFSSTPVLGYPSPPGGFSTGSPPGNTQQPLPGLPLASPPGVPPVSLHTQVQSVVPQQLLTVTAAPTAAPVTTTVTSQIQQVPVLLQPHFIKADSLLLTAMKTDGATVKAAGLSPLVSGTTVQTGPLPTLVSGGTILATVPLVVDAEKLPINRLAAGSKAPASAQSRGEKRTAHNAIEKRYRSSINDKIIELKDLVVGTEAKLNKSAVLRKAIDYIRFLQHSNQKLKQENLSLRTAVHKSKSLKDLVSACGSGGNTDVLMEGVKTEVEDTLTPPPSDAGSPFQSSPLSLGSRGSGSGGSGSDSEPDSPVFEDSKAKPEQRPSLHSRGMLDRSRLALCTLVFLCLSCNPLASLLGARGLPSPSDTTSVYHSPGRNVLGTESRDGPGWAQWLLPPVVWLLNGLLVLVSLVLLFVYGEPVTRPHSGPAVYFWRHRKQADLDLARGDFAQAAQQLWLALRALGRPLPTSHLDLACSLLWNLIRHLLQRLWVGRWLAGRAGGLQQDCALRVDASASARDAALVYHKLHQLHTMGKHTGGHLTATNLALSALNLAECAGDAVSVATLAEIYVAAALRVKTSLPRALHFLTRFFLSSARQACLAQSGSVPPAMQWLCHPVGHRFFVDGDWSVLSTPWESLYSLAGNPVDPLAQVTQLFREHLLERALNCVTQPNPSPGSADGDKEFSDALGYLQLLNSCSDAAGAPAYSFSISSSMATTTGVDPVAKWWASLTAVVIHWLRRDEEAAERLCPLVEHLPRVLQESERPLPRAALHSFKAARALLGCAKAESGPASLTICEKASGYLQDSLATTPASSSIDKAVQLFLCDLLLVVRTSLWRQQQPPAPAPAAQGTSSRPQASALELRGFQRDLSSLRRLAQSFRPAMRRVFLHEATARLMAGASPTRTHQLLDRSLRRRAGPGGKGGAVAELEPRPTRREHAEALLLASCYLPPGFLSAPGQRVGMLAEAARTLEKLGDRRLLHDCQQMLMRLGGGTTVTSS

Human SREBP1 cDNA (SEQ ID NO: 68)

atggacgagccacccttcagcgaggcggctttggagcaggcgctgggcgagccgtgcgatctggacgcggcgctgctgaccgacatcgaagacatgcttcagcttatcaacaaccaagacagtgacttccctggcctatttgacccaccctatgctgggagtggggcagggggcacagaccctgccagccccgataccagctccccaggcagcttgtctccacctcctgccacattgagctcctctcttgaagccttcctgagcgggccgcaggcagcgccctcacccctgtcccctccccagcctgcacccactccattgaagatgtacccgtccatgcccgctttctcccctgggcctggtatcaaggaagagtcagtgccactgagcatcctgcagacccccaccccacagcccctgccaggggccctcctgccacagagcttcccagccccagccccaccgcagttcagctccacccctgtgttaggctaccccagccctccgggaggcttctctacaggaagccctcccgggaacacccagcagccgctgcctggcctgccactggcttccccgccaggggtcccgcccgtctccttgcacacccaggtccagagtgtggtcccccagcagctactgacagtcacagctgcccccacggcagcccctgtaacgaccactgtgacctcgcagatccagcaggtcccggtcctgctgcagccccacttcatcaaggcagactcgctgcttctgacagccatgaagacagacggagccactgtgaaggcggcaggtctcagtcccctggtctctggcaccactgtgcagacagggcctttgccgaccctggtgagtggcggaaccatcttggcaacagtcccactggtcgtagatgcggagaagctgcctatcaaccggctcgcagctggcagcaaggccccggcctctgcccagagccgtggagagaagcgcacagcccacaacgccattgagaagcgctaccgctcctccatcaatgacaaaatcattgagctcaaggatctggtggtgggcactgaggcaaagctgaataaatctgctgtcttgcgcaaggccatcgactacattcgctttctgcaacacagcaaccagaaactcaagcaggagaacctaagtctgcgcactgctgtccacaaaagcaaatctctgaaggatctggtgtcggcctgtggcagtggagggaacacagacgtgctcatggagggcgtgaagactgaggtggaggacacactgaccccacccccctcggatgctggctcacctttccagagcagccccttgtcccttggcagcaggggcagtggcagcggtggcagtggcagtgactcggagcctgacagcccagtctttgaggacagcaaggcaaagccagagcagcggccgtctctgcacagccggggcatgctggaccgctcccgcctggccctgtgcacgctcgtcttcctctgcctgtcctgcaaccccttggcctccttgctgggggcccgggggcttcccagcccctcagataccaccagcgtctaccatagccctgggcgcaacgtgctgggcaccgagagcagagatggccctggctgggcccagtggctgctgcccccagtggtctggctgctcaatgggctgttggtgctcgtctccttggtgcttctctttgtctacggtgagccagtcacacggccccactcaggccccgccgtgtacttctggaggcatcgcaagcaggctgacctggacctggcccggggagactttgcccaggctgcccagcagctgtggctggccctgcgggcactgggccggcccctgcccacctcccacctggacctggcttgtagcctcctctggaacctcatccgtcacctgctgcagcgtctctgggtgggccgctggctggcaggccgggcagggggcctgcagcaggactgtgctctgcgagtggatgctagcgccagcgcccgagacgcagccctggtctaccataagctgcaccagctgcacaccatggggaagcacacaggcgggcacctcactgccaccaacctggcgctgagtgccctgaacctggcagagtgtgcaggggatgccgtgtctgtggcgacgctggccgagatctatgtggcggctgcattgagagtgaagaccagtctcccacgggccttgcattttctgacacgcttcttcctgagcagtgcccgccaggcctgcctggcacagagtggctcagtgcctcctgccatgcagtggctctgccaccccgtgggccaccgtttcttcgtggatggggactggtccgtgctcagtaccccatgggagagcctgtacagcttggccgggaacccagtggaccccctggcccaggtgactcagctattccgggaacatctcttagagcgagcactgaactgtgtgacccagcccaaccccagccctgggtcagctgatggggacaaggaattctcggatgccctcgggtacctgcagctgctgaacagctgttctgatgctgcgggggctcctgcctacagcttctccatcagttccagcatggccaccaccaccggcgtagacccggtggccaagtggtgggcctctctgacagctgtggtgatccactggctgcggcgggatgaggaggcggctgagcggctgtgcccgctggtggagcacctgccccgggtgctgcaggagtctgagagacccctgcccagggcagctctgcactccttcaaggctgcccgggccctgctgggctgtgccaaggcagagtctggtccagccagcctgaccatctgtgagaaggccagtgggtacctgcaggacagcctggctaccacaccagccagcagctccattgacaaggccgtgcagctgttcctgtgtgacctgcttcttgtggtgcgcaccagcctgtggcggcagcagcagcccccggccccggccccagcagcccagggcaccagcagcaggccccaggcttccgcccttgagctgcgtggcttccaacgggacctgagcagcctgaggcggctggcacagagcttccggcccgccatgcggagggtgttcctacatgaggccacggcccggctgatggcgggggccagccccacacggacacaccagctcctcgaccgcagtctgaggcggcgggcaggccccggtggcaaaggaggcgcggtggcggagctggagccgcggcccacgcggcgggagcacgcggaggccttgctgctggcctcctgctacctgccccccggcttcctgtcggcgcccgggcagcgcgtgggcatgctggctgaggcggcgcgcacactcgagaagcttggcgatcgccggctgctgcacgactgtcagcagatgctcatgcgcctgggcggtgggaccactgtcacttccagctag

Non-limiting examples of commercial ELISA assays that can be used to determine IFN-gamma expression levels are obtained from Andi biology (R & D Systems), siemens Feier technology (Thermo Fisher Scientific), ai Bokang (Abcam), eimer's (Enzo Life Sciences) and Rayoco (RayBiotech). Mature human IFN-gamma protein and cDNA sequences are shown below.

Mature human IFN-gamma (SEQ ID NO: 69)

Human IFN-gamma cDNA (SEQ ID NO: 70)

Non-limiting examples of commercial ELISA assays that can be used to determine the expression level of granzyme B are obtained from rapaone, sameifer's technology and animate organisms. Mature human granzyme B protein and cDNA sequences are shown below.

Mature human granzyme B (SEQ ID NO: 71)

Human granzyme B cDNA (SEQ ID NO: 72)

Non-limiting examples of commercial ELISA assays that can be used to determine MYC expression levels are available from Invitrogen life technologies (Invitrogen), LSBio, biocodon technologies company (Biocodon Technologies), and ELISA Genie. Mature human MYC protein and cDNA sequences are shown below.

Human Myc protein (SEQ ID NO: 329)

Human Myc cDNA (SEQ ID NO: 330)

cagaggagga acgagctaaa acggagcttt tttgccctgc gtgaccagat cccggagttggaaaacaatg aaaaggcccc caaggtagtt atccttaaaa aagccacagc atacatcctgtccgtccaag cagaggagca aaagctcatt tctgaagagg acttgttgcg gaaacgacgagaacagttga aacacaaact tgaacagcta cggaactctt gtgcgtaa

In some embodiments, activated NK cells (e.g., human activated NK cells) can exhibit an ability to kill senescent cells (e.g., any senescent cells described herein) in a subject (e.g., any of the subjects described herein) or in vitro as compared to resting NK cells (e.g., human resting NK cells) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, or at least 300%.

In some embodiments, the ability of an activated NK cell (e.g., a human activated NK cell) to kill senescent cells (e.g., any of the senescent cells described herein) in a subject (e.g., any of the subjects described herein) or in vivo can be improved by about 10% to about 500% (or any subrange of this range described herein) as compared to a resting NK cell (e.g., a human resting NK cell).

In some embodiments, an activated NK cell (e.g., a human activated NK cell) can exhibit enhanced (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, or at least 300%) cytotoxic activity in contact cytotoxicity assays in the presence of an antibody that specifically binds to an antigen present on a senescent or target cell as compared to a resting NK cell (e.g., a human resting NK cell).

In some embodiments, activated NK cells (e.g., human activated NK cells) may exhibit enhanced (e.g., enhanced by about 10% to about 500%, or any subrange of this range described herein) cytotoxic activity in contact with a cytotoxicity test in the presence of antibodies that specifically bind to antigens present on senescent or target cells as compared to resting NK cells (e.g., human resting NK cells).

In some embodiments, activated NK cells can be produced by a method comprising: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are haploid resting NK cells. In some embodiments of these methods, the resting NK cells are allogeneic resting NK cells. In some embodiments of these methods, the resting NK cells are artificial NK cells. In some embodiments of any of these methods, the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

In some embodiments of these methods, the liquid medium is a serum-free liquid medium. In some embodiments of any one of the methods described herein, the liquid medium is a chemically-defined liquid medium. Some embodiments of these methods further comprise isolating the activated NK cells (and optionally further administering a therapeutically effective amount of the activated NK cells to a subject, such as any of the subjects described herein).

In some embodiments of these methods, the contacting step is performed for a period of time ranging from about 2 hours to about 20 days (e.g., about 2 hours to about 18 days, about 2 hours to about 16 days, about 2 hours to about 14 days, about 2 hours to about 12 days, about 2 hours to about 10 days, about 2 hours to about 8 days, about 2 hours to about 7 days, about 2 hours to about 6 days, about 2 hours to about 5 days, about 2 hours to about 4 days, about 2 hours to about 3 days, about 2 hours to about 2 days, about 2 hours to about 1 day, about 6 hours to about 18 days, about 6 hours to about 16 days, about 6 hours to about 14 days, about 6 hours to about 12 days, about 6 hours to about 10 days, about 6 hours to about 8 days, about 6 hours to about 7 days, about 6 hours to about 6 days, about 6 hours to about 5 days, about 6 hours to about 4 days, about 6 hours to about 3 days, about 6 hours to about 2 days, about 6 hours to about 1 day, about 12 hours to about 18 days, about 12 days, about 6 hours to about 18 days from about 12 hours to about 16 days, from about 12 hours to about 14 days, from about 12 hours to about 12 days, from about 12 hours to about 10 days, from about 12 hours to about 8 days, from about 12 hours to about 7 days, from about 12 hours to about 6 days, from about 12 hours to about 5 days, from about 12 hours to about 4 days, from about 12 hours to about 3 days, from about 12 hours to about 2 days, from about 12 hours to about 1 day, from about 1 day to about 18 days, from about 1 day to about 16 days, from about 1 day to about 15 days, from about 1 day to about 14 days, from about 1 day to about 12 days, from about 1 day to about 10 days, from about 1 day to about 8 days, from about 1 day to about 7 days, from about 1 day to about 6 days, from about 1 day to about 5 days, from about 1 day to about 4 days, from about 1 day to about 3 days, from about 1 day to about 2 days, from about 2 days to about 18 days, from about 2 days to about 16 days, from about 2 days, from about 14 days, about 1 day to about 10 days, about 1 day, about 5 days, about 1 day to about 5 days, and about 5 days, about 2 to about 12 days, about 2 to about 10 days, about 2 to about 8 days, about 2 to about 7 days, about 2 to about 6 days, about 2 to about 5 days, about 2 to about 4 days, about 2 to about 3 days, about 3 to about 18 days, about 3 to about 16 days, about 3 to about 14 days, about 3 to about 12 days, about 3 to about 10 days, about 3 to about 8 days, about 3 to about 7 days, about 3 to about 6 days, about 3 to about 5 days, about 3 to about 4 days, about 4 to about 18 days, about 4 to about 16 days, about 4 to about 14 days, about 4 to about 12 days, about 4 to about 10 days, about 4 to about 8 days, about 4 to about 7 days, about 4 to about 6 days, about 4 to about 5 days, about 5 to about 18 days, about 5 to about 16 days, about 5 to about 14 to about 5 days, about 5 to about 10 days about 5 to about 8 days, about 5 to about 7 days, about 5 to about 6 days, about 6 to about 18 days, about 6 to about 16 days, about 6 to about 14 days, about 6 to about 12 days, about 6 to about 10 days, about 6 to about 8 days, about 6 to about 7 days, about 7 to about 18 days, about 7 to about 16 days, about 7 to about 14 days, about 7 to about 12 days, about 7 to about 10 days, about 7 to about 8 days, about 8 to about 18 days, about 8 to about 16 days, about 8 to about 14 days, about 8 to about 12 days, about 8 to about 10 days, about 9 to about 18 days, about 9 to about 16 days, about 9 to about 14 days, about 9 to about 12 days, about 12 to about 18 days, about 12 to about 16 days, about 12 to about 14 days, about 14 to about 18 days, about 14 to about 16 days, or about 16 to about 16 days.

NK cell activators

The methods provided herein comprise the use or administration of one or more NK cell activators. In some embodiments, the NK cell activator may be a protein. In some embodiments, the NK cell activator can be a single chain chimeric polypeptide (e.g., any of the single chain chimeric polypeptides described herein), a multi-chain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein, such as the exemplary type a and type B multi-chain chimeric polypeptides described herein), an antibody, a recombinant cytokine, or an interleukin (e.g., any of the recombinant cytokines or interleukins described herein), and a soluble interleukin or cytokine receptor (e.g., any of the soluble interleukins or cytokine receptors described herein). In some embodiments, the NK cell activator may be a small molecule (e.g., a liver glucose synthase kinase-3 (GSK 3) inhibitor, such as CHIR99021 described in Cichocki et al, cancer Res.77:5664-5675, 2017) or an aptamer.

In some embodiments of any of the one or more NK cell activators provided herein, at least one of the one or more NK cell activators causes activation of one or more (e.g., two, three, four, five, six, seven, or eight) of: IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor, IL-33 receptor, CD16, CD69, CD25, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR DS1, KIR2Ds2/3, KIR2DL4, KIR2DS5 and KIR3DS1 (e.g., in immune cells such as human immune cells, e.g., human NK cells).

In some embodiments, at least one of the one or more NK cell activators that cause activation of the IL-2 receptor is soluble IL-2 or an agonistic antibody that specifically binds to the IL-2 receptor.

In some embodiments, at least one of the one or more NK cell activators that cause activation of the IL-7 receptor is soluble IL-7 or an agonistic antibody that specifically binds to the IL-7 receptor.

In some embodiments, one or more causes IL-12 receptor activation of NK cell activator in at least one is soluble IL-12 or specific binding to IL-12 receptor agonistic antibody.

In some embodiments, at least one of the one or more NK cell activators that cause activation of the IL-15 receptor is soluble IL-15 or an agonistic antibody that specifically binds to the IL-15 receptor.

In some embodiments, at least one of the one or more NK cell activators that cause activation of the IL-21 receptor is soluble IL-21 or an agonistic antibody that specifically binds to the IL-21 receptor.

In some embodiments, at least one of the one or more NK cell activators that cause activation of the IL-33 receptor is soluble IL-33 or an agonistic antibody that specifically binds to the IL-33 receptor.

In some embodiments, at least one of the one or more NK cell activators that cause activation of CD16 is an agonistic antibody that specifically binds to CD 16.

In some embodiments, at least one of the one or more NK cell activators that cause CD69 activation is an agonistic antibody that specifically binds to CD 69.

In some embodiments, at least one of the one or more NK cell activators that cause activation of CD25, CD59 is an agonistic antibody that specifically binds to CD25, CD 59.

In some embodiments, at least one of the one or more NK cell activators that cause activation of CD352 is an agonistic antibody that specifically binds to CD 352.

In some embodiments, at least one of the one or more NK cell activators that cause NKp80 activation is an agonistic antibody that specifically binds to NKp 80.

In some embodiments, at least one of the one or more NK cell activators that cause DNAM-1 activation is an agonistic antibody that specifically binds to DNAM-1.

In some embodiments, at least one of the one or more NK cell activators that cause activation of 2B4 is an agonistic antibody that specifically binds to 2B 4.

In some embodiments, at least one of the one or more NK cell activators that cause NKp30 activation is an agonistic antibody that specifically binds to NKp 30.

In some embodiments, at least one of the one or more NK cell activators that cause activation of NKp44 is an agonistic antibody that specifically binds to NKp 44.

In some embodiments, at least one of the one or more NK cell activators that cause activation of NKp46 is an agonistic antibody that specifically binds to NKp 46.

In some embodiments, at least one of the one or more NK cell activators that cause NKG2D activation is an agonistic antibody that specifically binds to NKG 2D.

In some embodiments, at least one of the one or more NK cell activators that cause activation of KIR2DS1 is an agonistic antibody that specifically binds to KIT2DS 1.

In some embodiments, at least one of the one or more NK cell activators that cause KIR2DS2/3 activation is an agonistic antibody that specifically binds to KIT2DS 2/3.

In some embodiments, at least one of the one or more NK cell activators that cause KIR2DL4 activation is an agonistic antibody that specifically binds to KIT2DL 4.

In some embodiments, at least one of the one or more NK cell activators that cause activation of KIR2DS4 is an agonistic antibody that specifically binds to KIT2DS 4.

In some embodiments, at least one of the one or more NK cell activators that cause activation of KIR2DS5 is an agonistic antibody that specifically binds to KIT2DS 5.

In some embodiments, at least one of the one or more NK cell activators that cause activation of KIR3DS1 is an agonistic antibody that specifically binds to KIT3DS 1.

In some embodiments of any one of the one or more NK cell activators provided herein, at least one (e.g., two, three, four, or five) of the one or more NK cell activators causes a decrease in activation of one or more of the following compared to the level of activation in the absence of the one or more NK cell activators: PD-1, TGF-beta receptor, TIGIT, CD1, TIM-3, siglec-7, IRP60, tactive, IL1R8, NKG2A/KLRD1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2 (e.g., in immune cells such as human immune cells, e.g., human NK cells).

In some embodiments, at least one of the one or more NK cell activators that result in reduced activation of the TGF- β receptor is a soluble TGF- β receptor, an antibody that specifically binds to TGF- β, or an antagonistic antibody that specifically binds to the TGF- β receptor.

In some embodiments, at least one of the one or more NK cell activators that result in reduced TIGIT activation is an antagonistic antibody that specifically binds to TIGIT, a soluble TIGIT, or an antibody that specifically binds to TIGIT ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced activation of CD1 is an antagonistic antibody that specifically binds to CD1, soluble CD1, or an antibody that specifically binds to a CD1 ligand.

For some embodiments, at least one of the one or more NK cell activators that result in reduced TIM-3 activation is an antagonistic antibody that specifically binds to TIM-3, soluble TIM-3, or an antibody that specifically binds to a TIM-3 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced activation of Siglec-7 is an antagonistic antibody that specifically binds to Siglec-7, a soluble Siglec-7, or an antibody that specifically binds to a Siglec-7 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced IRP-60 activation is an antagonistic antibody that specifically binds to IRP-60, a soluble IRP-60, or an antibody that specifically binds to an IRP-60 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced activation of Tactile is an antagonistic antibody that specifically binds to Tactile, a soluble Tactile, or an antibody that specifically binds to a Tactile ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced activation of IL1R8 is an antagonistic antibody that specifically binds to IL1R8, soluble IL1R8, or an antibody that specifically binds to an IL1R8 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced activation of NKG2A/KLRD1 is an antagonistic antibody that specifically binds to NKG2A/KLRD1, a soluble NKG2A/KLRD1, or an antibody that specifically binds to a NKG2A/KLRD1 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced KIR2DL1 activation is an antagonistic antibody that specifically binds to KIR2DL1, a soluble KIR2DL1, or an antibody that specifically binds to a KIR2DL1 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced KIR2DL2/3 activation is an antagonistic antibody that specifically binds to KIR2DL2/3, a soluble KIR2DL2/3, or an antibody that specifically binds to a KIR2DL2/3 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced KIR2DL5 activation is an antagonistic antibody that specifically binds to KIR2DL5, a soluble KIR2DL5, or an antibody that specifically binds to a KIR2DL5 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced KIR3DL1 activation is an antagonistic antibody that specifically binds to KIR3DL1, a soluble KIR3DL1, or an antibody that specifically binds to a KIR3DL1 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced KIR3DL2 activation is an antagonistic antibody that specifically binds to KIR3DL2, a soluble KIR3DL2, or an antibody that specifically binds to a KIR3DL2 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced activation of ILT2/LIR-1 is an antagonistic antibody that specifically binds to ILT2/LIR-1, a soluble ILT2/LIR-1, or an antibody that specifically binds to a ILT2/LIR-1 ligand.

In some embodiments, at least one of the one or more NK cell activators that result in reduced LAG2 activation is an antagonistic antibody that specifically binds to LAG2, soluble LAG2, or an antibody that specifically binds to a LAG2 ligand.

Non-limiting examples of NK cell activators are described below and may be used in any combination.

For some embodiments, the NK cell activator may be soluble PD-1, soluble PD-L1, soluble TIGIT, soluble CD1, or soluble TIM-3. Non-limiting examples of soluble PD-1, PD-L1, TIGIT, CD1 and TIM-3 are provided below.

Human soluble PD-1 (SEQ ID NO: 73)

Human soluble PD-L1 (SEQ ID NO: 74)

Human soluble TIGIT (SEQ ID NO: 75)

Human soluble CD1A (SEQ ID NO: 76)

Human soluble TIM3 (SEQ ID NO: 77)

In some embodiments, the soluble PD-1 protein may comprise a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO 73.

In some embodiments, the soluble PD-L1 protein may comprise a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO 74.

In some embodiments, the soluble TIGIT protein may comprise a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO.

In some embodiments, the soluble CD1A protein may comprise a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO 76.

For some embodiments, the soluble TIM3 protein may comprise a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO 77.

Recombinant antibodies

In some embodiments, the NK activator may be: agonistic antibodies that specifically bind to the IL-2 receptor (see, e.g., those described in Gaulton et al, clinical Immunology and Immunopathology 36 (1): 18-29, 1985), agonistic antibodies that specifically bind to the IL-7 receptor, agonistic antibodies that specifically bind to the IL-12 receptor (see, e.g., those described in Rogge et al, J.Immunol.162 (7): 3926-3932, 1999), agonistic antibodies that specifically bind to the IL-15 receptor, agonistic antibodies that specifically bind to the IL-21 receptor (see, e.g., those described in U.S. patent application publication No. 2006/159555), agonistic antibodies that specifically bind to the IL-33 receptor (see, e.g., those described in U.S. patent application publication No. 2007/160579), antagonistic antibodies that specifically bind to PD-1 (see, e.g., those described in U.S. patent No. 7,521,051), antibodies that specifically bind to PD-L1 (see, e.g., those described in U.S. patent application No. 8,217,149), agonistic antibodies that specifically bind to the IL-15 receptor (see, e.g., those described in WO 20147), agonistic antibodies that specifically bind to the IL-33 receptor (see, e.g., those described in U.S. patent application publication No. 2006/159755), agonistic antibodies that specifically bind to the TGF-1 receptor (see, e.g., those that specifically bind to the TGF-specific binding to the receptor, for example, szalay et al, j.immunol.162 (12): 6955-6958, 1999), antibodies that specifically bind to CD1 ligands (see, e.g., kain et al, immunity 41 (4): 543-554, 2014), antagonistic antibodies that specifically bind to TIM-3 (see, e.g., those described in U.S. patent application publication No. 2015/218274), antibodies that specifically bind to TIM-3 ligand (see, e.g., those described in U.S. patent application publication No. 2017/283499), agonistic antibodies that specifically bind to CD69 (see, e.g., moretta et al, journal of Experimental Medicine 174): 1393 1991), agonistic antibodies that specifically bind to CD25, CD59, agonistic antibodies that specifically bind to CD352 (see, e.g., yifit et al, oncotarget 7:26346-26360, 2016), agonistic antibodies that specifically bind to NKp80 (see, e.g., peipp et al, oncotarget 6:32075-32088, 2015), agonistic antibodies that specifically bind to DNAM-1, agonistic antibodies that specifically bind to 2B4 (see, e.g., sandusky et al, european j. Immunol.36:3268-3276, 2006), agonistic antibodies that specifically bind to NKp30 (see, e.g., kellner et al, oncoImmunology 5:1-12, 2016), agonistic antibodies that specifically bind to NKp44, agonistic antibodies that specifically bind to NKp46 (see, e.g., xiong et al, J.Clin. Invest.123:4264-4272, 2013), agonistic antibodies that specifically bind to NKG2D (see, e.g., kellner et al, oncoImmunology 5:1-12, 2016), agonistic antibodies that specifically bind to KIR2DS1 (see, e.g., xiong et al, j.clin.invest.123:4264-4272, 2013), agonistic antibodies that specifically bind to KIR2Ds2/3 (see, e.g., borgerding et al, exp. Therapeutics 38:213-221, 2010), agonistic antibodies that specifically bind to KIR2DL4 (see, e.g., miah et al, j.immunol.180:2922-32, 2008), agonistic antibodies that specifically bind to KIR2DS4 (see, e.g., czaja et al, genes and Immunity: 33-37, 2014), agonistic antibodies that specifically bind to KIR2DS5 (see, e.g., czaja et al, genes and Immunity: 33-37, 2014), agonistic antibodies that specifically bind to KIR3DS1 (see, e.g., czaja et al, genes and Immunity: 33-37, 2014), antagonistic antibodies that specifically bind to Siglec-7 (see, e.g., hudak et al, nature Chemical Biology 10:69-75, 2014), antagonistic antibodies that specifically bind to IRP60 (see, e.g., bachelet et al, j. Biol. Chem.281:27190-27196, 2006), antagonistic antibodies that specifically bind to Tactile (see, e.g., brooks et al, eur. J. Cancer 61 (suppl. 1): those described in S189, 2016), antagonistic antibodies that specifically bind to IL1R8 (see, e.g., molgora et al, front immunol.7:1,2016), antagonistic antibodies that specifically bind to NKG2A/KLRD1 (see, e.g., kim et al Infection Immunity 76:5873-5882, 2008), antagonistic antibodies that specifically bind to KIR2DL1 (see, e.g., weiner et al, cell 148:1081-1084, 2012), antagonistic antibodies that specifically bind to KIR2DL2/3 (see, e.g., weiner et al, cell 148:1081-1084, 2012), antagonistic antibodies that specifically bind to KIR2DL5 (see, e.g., those described in US 9,067,997), and antagonistic antibodies that specifically bind to KIR3DL1 (see, e.g., those described in US 9,067,997), antagonistic antibodies that specifically bind to KIR3DL2 (see, e.g., those described in US 9,067,997), antagonistic antibodies that specifically bind to ILT2/LIR-1 (see, e.g., those described in US 8,133,485), and antagonistic antibodies that specifically bind to LAG-2.

Recombinant antibodies as NK cell activators may be any of the exemplary types of antibodies (e.g., human or humanized) or any of the exemplary antibody fragments described herein. Recombinant antibodies that are NK cell activators may include, for example, any of the antigen binding domains described herein.

Recombinant interleukins or cytokines

In some embodiments, NK activators may be, for example, soluble IL-2, soluble IL-7, soluble IL-12, soluble IL-15, soluble IL-21, and soluble IL-33. Non-limiting examples of soluble IL-12, IL-15, IL-21 and IL-33 are provided below.

Human soluble IL-2 (SEQ ID NO: 78)

Human soluble IL-7 (SEQ ID NO: 79)

Human soluble IL-12 subunit alpha (SEQ ID NO: 80)

Human soluble IL-12 subunit beta (SEQ ID NO: 81)

Human soluble IL-15 (SEQ ID NO: 82)

Human soluble IL-21 (SEQ ID NO: 83)

Human soluble IL-33 (SEQ ID NO: 84)

In some embodiments, the soluble IL-2 protein may include a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO. 78.

In some embodiments, the soluble IL-7 protein may include a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO 79.

In some embodiments, the soluble IL-2 protein comprises a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO; and a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO. 81.

In some embodiments, the soluble IL-15 protein may include a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO 82.

In some embodiments, the soluble IL-21 protein may include a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO. 83.

In some embodiments, the soluble IL-33 protein may include a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO.

Soluble cytokine or interleukin receptor

In some embodiments of any of the soluble cytokines or interleukin receptors described herein, the soluble cytokine or interleukin receptor may be a soluble TGF- β receptor. In some embodiments, the soluble TGF-beta receptor is soluble TGF-beta receptor I (TGF-beta RI) (see, e.g., docagne et al Journal of Biological Chemistry 276 (49): 46243-46250, 2001), soluble TGF-beta receptor II (TGF-beta RII) (see, e.g., yung et al, am. J. Resp. Crit. Care Med.194 (9): 1140-1151, 2016), soluble TGF-beta RIII (see, e.g., heng et al, plamenta 57:320, 2017). In some embodiments, the soluble TGF-beta receptor is a receptor "trap" for TGF-beta (see, e.g., zwaagstra et al, mol. Cancer Ther.11 (7): 1477-1487, 2012; and De Crescenzo et al, transforming Growth Factor-beta in Cancer Therapy, volume II, pp 671-684).

Other embodiments of soluble cytokines or soluble interleukin receptors are known in the art.

Single chain chimeric polypeptides

Non-limiting examples of NK cell activators are single chain chimeric polypeptides comprising: (i) a first target binding domain (e.g., any of the target binding domains described herein or known in the art), (ii) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art), and (iii) a second target binding domain (e.g., any of the target binding domains described herein or known in the art).

In some embodiments of any of the single-chain chimeric polypeptides described herein, the total length of the single chain chimeric polypeptide can be from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, from about 50 amino acids to about 700 amino acids, from about 50 amino acids to about 650 amino acids, from about 50 amino acids to about 600 amino acids, from about 50 amino acids to about 550 amino acids, from about 50 amino acids to about 500 amino acids, from about 50 amino acids to about 480 amino acids, from about 50 amino acids to about 460 amino acids, from about 50 amino acids about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids about 100 amino acids to about 360 amino acids, about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about, about 150 to about 800 amino acids, about 150 to about 750 amino acids, about 150 to about 700 amino acids, about 150 to about 650 amino acids, about 150 to about 600 amino acids, about 150 to about 550 amino acids, about 150 to about 500 amino acids, about 150 to about 480 amino acids, about 150 to about 460 amino acids, about 150 to about 440 amino acids, about 150 to about 420 amino acids, about 150 to about 400 amino acids, about 150 to about 380 amino acids, about 150 to about 360 amino acids, about 150 to about 340 amino acids, about 150 to about 320 amino acids, about 150 to about 300 amino acids, about 150 to about 280 amino acids, about 150 to about 260 amino acids about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids, about 200 amino acids to about 480 amino acids, about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids about 220 to about 950 amino acids, about 220 to about 900 amino acids, about 220 to about 850 amino acids, about 220 to about 800 amino acids, about 220 to about 750 amino acids, about 220 to about 700 amino acids, about 220 to about 650 amino acids, about 220 to about 600 amino acids, about 220 to about 550 amino acids, about 220 to about 500 amino acids, about 220 to about 480 amino acids, about 220 to about 460 amino acids, about 220 to about 440 amino acids, about 220 to about 420 amino acids, about 220 to about 400 amino acids, about 220 to about 380 amino acids, about 220 to about 360 amino acids, about 220 to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 to about 950 amino acids, about 260 to about 900 amino acids, about 260 to about 850 amino acids, about 260 to about 800 amino acids, about 260 to about 750 amino acids, about 260 to about 700 amino acids, about 260 to about 650 amino acids, about 260 to about 600 amino acids, about 260 to about 550 amino acids, about 260 to about 500 amino acids, about 260 to about 480 amino acids, about 260 to about 460 amino acids, about 260 to about 440 amino acids, about 260 to about 420 amino acids, about 260 to about 400 amino acids, about 260 to about 380 amino acids, about 260 to about 360 amino acids, about 260 to about 340 amino acids, about 260 to about 320 amino acids about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids, about 320 amino acids to about 340 amino acids about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440 amino acids, about 420 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 700 amino acids, about 1000 amino acids, about, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids, about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids.

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are directly adjacent to each other. In some embodiments of any one of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any one of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any one of the exemplary first target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any one of the exemplary soluble tissue factor domains described herein). In some embodiments of any of the single-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are directly adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

In some embodiments of any of the single-chain chimeric polypeptides described herein, a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are directly adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art). In some embodiments of any of the single-chain chimeric polypeptides described herein, the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are directly adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art).

In some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the following sequence:

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(SEQ ID NO:85)。

in some embodiments, the single-chain chimeric polypeptide is encoded by a nucleic acid comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the sequence:

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG(SEQ ID NO:86)。

in some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the following sequence:

MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(SEQ ID NO:87)

in some embodiments, the single-chain chimeric polypeptide is encoded by a nucleic acid comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the sequence:

ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCCCAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG(SEQ ID NO:88)。

in some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the following sequence:

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(SEQ ID NO:89)。

In some embodiments, the single-chain chimeric polypeptide is encoded by a nucleic acid comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the sequence:

GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAGCAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC(SEQ ID NO:90)。

in some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the following sequence:

MKWVTFISLLFLFSSAYSVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(SEQ ID NO:91)。

in some embodiments, the single-chain chimeric polypeptide is encoded by a nucleic acid comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the sequence:

ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCTACAGCGTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGGAGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAGCAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC(SEQ ID NO:92)。

some embodiments of any of the single-chain chimeric polypeptides described herein can further comprise one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the N-terminus and/or C-terminus thereof.

In some embodiments, a single-chain chimeric polypeptide may include one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at its N-terminus. In some embodiments, one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the N-terminus of the single-chain chimeric polypeptide can directly abut the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any one of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide and the first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any one of the exemplary soluble tissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at its C-terminus. In some embodiments, one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the C-terminus of the single-chain chimeric polypeptide directly abuts the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at its N-terminus and its C-terminus. In some embodiments, one of the one or more additional antigen binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the N-terminus of the single-chain chimeric polypeptide directly adjoins the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains). In some embodiments, one of the one or more additional antigen binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the C-terminus directly adjoins the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the or each tissue factor domain (e.g., any of the exemplary soluble tissue factor domains). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein).

In some embodiments of any of the single-chain chimeric polypeptides described herein, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) specifically bind to the same antigen. In some embodiments, two or more (e.g., three, four, five, six, seven, eight, nine, or ten) of the first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) specifically bind to the same epitope. In some embodiments, the first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) comprise the same amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptides described herein, a first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), a second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) each specifically bind to the same antigen. In some embodiments, a first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), a second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains each comprise the same amino acid sequence.

In some embodiments of any of the single-chain chimeric polypeptides described herein, a first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), a second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) specifically bind to different antigens.

In some embodiments of any one of the single-chain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains (e.g., any of the exemplary antigen binding domains described herein or known in the art). In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each an antigen binding domain (e.g., any of the exemplary antigen binding domains described herein or known in the art). In some embodiments, the antigen binding domain may comprise an scFv or a single domain antibody.

In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC A, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-. Beta.receptor II (TGF-. Beta.RII), ligands of DNAM1, ligands of NKp46, ligands of NKp44 a ligand for NKG2D, a ligand for NKp30, a ligand for scmhc i, a ligand for scmhc II, a ligand for scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD 28.

In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine protein. Non-limiting examples of soluble interleukin proteins and soluble cytokine proteins include: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine receptor. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include: soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble NKG2D, soluble NKP30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3 or soluble CD28.

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the target binding domains described herein), the second target binding domain (e.g., any of the target binding domains described herein), and one or more additional target binding domains (e.g., any of the target binding domains described herein) can each independently specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands for TGF-beta receptor II (TGF-beta RII) a ligand for TGF- βriii, a ligand for DNAM1, a ligand for NKp46, a ligand for NKp44, a ligand for NKG2D, a ligand for NKp30, a ligand for scmhc i, a ligand for scTCR, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, and a receptor for CD 122.

In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine protein. In some embodiments of any one of the multi-chain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

A-type multi-chain chimeric polypeptides

A non-limiting example of an NK cell activator is a multi-chain chimeric polypeptide comprising: (a) A first chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain in a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) A second domain in a pair of affinity domains and (ii) a second target binding domain, wherein the first chimeric polypeptide associates with the second chimeric polypeptide via binding of the first domain to the second domain in the pair of affinity domains.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the total length of the first multi-chain chimeric polypeptide and/or the second chimeric polypeptide can each independently be from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, from about 50 amino acids to about 700 amino acids, from about 50 amino acids to about 650 amino acids, from about 50 amino acids to about 600 amino acids, from about 50 amino acids to about 550 amino acids, from about 50 amino acids to about 500 amino acids, from about 50 amino acids to about 480 amino acids about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 50 amino acids to about 280 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids, about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 100 amino acids, about 150 to about 850 amino acids, about 150 to about 800 amino acids, about 150 to about 750 amino acids, about 150 to about 700 amino acids, about 150 to about 650 amino acids, about 150 to about 600 amino acids, about 150 to about 550 amino acids, about 150 to about 500 amino acids, about 150 to about 480 amino acids, about 150 to about 460 amino acids, about 150 to about 440 amino acids, about 150 to about 420 amino acids, about 150 to about 400 amino acids, about 150 to about 380 amino acids, about 150 to about 360 amino acids, about 150 to about 340 amino acids, about 150 to about 320 amino acids, about 150 to about 300 amino acids, about 150 to about 280 amino acids about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids, about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids about 220 to about 1000 amino acids, about 220 to about 950 amino acids, about 220 to about 900 amino acids, about 220 to about 850 amino acids, about 220 to about 800 amino acids, about 220 to about 750 amino acids, about 220 to about 700 amino acids, about 220 to about 650 amino acids, about 220 to about 600 amino acids, about 220 to about 550 amino acids, about 220 to about 500 amino acids, about 220 to about 480 amino acids, about 220 to about 460 amino acids, about 220 to about 440 amino acids, about 220 to about 420 amino acids, about 220 to about 400 amino acids, about 220 to about 380 amino acids, about 220 to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 280 amino acids, about 320 amino acids to about 280 amino acids, about 260 amino acids to about 280 amino acids, about 300 amino acids to about 280 amino acids, about 260 to about 280 amino acids, about 300 amino acids to about 280 amino acids, about 300 amino acids to about 280, about 280 amino acids to about 280 amino acids, about 260 amino acids to about 280, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids about 320 amino acids to about 340 amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 440 amino acids, about 420 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 750 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids, about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) in the first chimeric polypeptide.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain in the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein) are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) in the first chimeric polypeptide and the first domain in the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of a pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pair of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are directly contiguous with each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein,

the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between a second domain of a pair of affinity domains in the second chimeric polypeptide (e.g., any of the exemplary pair of affinity domains described herein or known in the art) and a second target binding domain (e.g., any of the exemplary second target binding domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art), wherein at least one of the one or more additional antigen binding domains is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary pair of affinity domains described herein). In some embodiments, the first chimeric polypeptide may further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art), and/or at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein) between the at least one of the one or more additional target binding domains (e.g., any of the exemplary pair of affinity domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains located at the N-terminus and/or the C-terminus of the first chimeric polypeptide. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) directly abuts a first domain in a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pair of affinity domains described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and a first domain in a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pair of affinity domains described herein). In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) directly adjoins the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is disposed at the N-and/or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein). In some embodiments, in the first chimeric polypeptide, at least one additional target binding domain of the one or more additional target binding domains disposed at the N-terminus (e.g., any of the exemplary target binding domains described herein or known in the art) directly abuts the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed in the first chimeric polypeptide between at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein). In some embodiments, in the first chimeric polypeptide, at least one additional target binding domain of the one or more additional target binding domains disposed at the C-terminus (e.g., any of the exemplary target binding domains described herein or known in the art) directly abuts the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed in the first chimeric polypeptide between at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the first domains described herein or any of the exemplary pair of affinity domains described herein) directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between (i) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein) and at least one of (e.g., any of the exemplary target binding domains described herein or known in the art) of one or more additional target binding domains, at least one of which is located between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of an exemplary pair of affinity domains described herein), and/or (ii) at least one of the binding domains of the one or more target binding domains of the one or more additional target binding domains, at least one of which is located between the one or more additional binding domains of the one or more target binding domains and the one or more additional binding domains of the soluble tissue factor domains.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the N-terminus and/or C-terminus of the second chimeric polypeptide. In some embodiments, in the second chimeric polypeptide, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) directly abuts a second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pair of affinity domains described herein). In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) in the second chimeric polypeptide between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and a second domain in a pair of affinity domains (e.g., any of the exemplary second domains of the exemplary pair of affinity domains described herein). In some embodiments, in the second chimeric polypeptide, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) directly adjoins the second target binding domain (e.g., any of the target binding domains described herein or known in the art). In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and one or more additional target binding domains comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to different antigens. In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each an antigen binding domain (e.g., scFv or single domain antibody).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any one of the exemplary target binding domains described herein or known in the art) specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC A, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-. Beta.receptor II (TGF-. Beta.RII), ligands of DNAM1, ligands of NKp46, ligands of NKp44 a ligand for NKG2D, a ligand for NKp30, a ligand for scmhc i, a ligand for scmhc II, a ligand for scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD 28.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine protein. Non-limiting examples of soluble interleukin proteins and soluble cytokine proteins include: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine receptor. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include: soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble NKG2D, soluble NKP30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3 or soluble CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the target binding domains described herein), the second target binding domain (e.g., any of the target binding domains described herein), and one or more additional target binding domains (e.g., any of the target binding domains described herein) can each independently specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, TGF-beta receptor II (TGF-. Beta.RII) ligand, TGF-. Beta.RIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, PDGF-DD receptor, receptor (SCF-DD receptor), receptor (receptor 3, MICP receptor) receptor of MIC-3, receptor-receptor (CD-B receptor) 122, receptor-3 of MIC-cell-like receptor (receptor 3, receptor-A of MIC-receptor-3.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain (e.g., any of the target binding domains described herein), the second target binding domain (e.g., any of the target binding domains described herein), and one or more additional binding domains (e.g., any of the target binding domains described herein) is a soluble interleukin or cytokine protein. In some embodiments of any one of the multi-chain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor or cytokine receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

B-type multi-chain chimeric polypeptides

A non-limiting example of an NK cell activator is a multi-chain chimeric polypeptide comprising: (a) first and second chimeric polypeptides, each comprising: (i) a first target binding domain, (ii) an Fc domain, and (iii) a first domain of a pair of affinity domains; and (b) third and fourth chimeric polypeptides, each comprising: (i) A second domain in a pair of affinity domains, and (ii) a second target binding domain, wherein the first and second chimeric polypeptides associate with the third and fourth chimeric polypeptides via binding of the first domain to the second domain in the pair of affinity domains, and the first and second chimeric polypeptides associate via their Fc domains.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the Fc domain (e.g., any of the exemplary Fc domains described herein) are directly adjacent to each other in the first and second chimeric polypeptides. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first and second chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the Fc domain (e.g., any of the exemplary Fc domains described herein) in the first and second chimeric polypeptides.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, an Fc domain (e.g., any of the exemplary Fc domains described herein) is directly contiguous with a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein) in the first and second chimeric polypeptides. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first and second chimeric polypeptides further comprise a linker sequence (e.g., any one of the exemplary linker sequences described herein or known in the art) between the Fc domain in the first and second chimeric polypeptides (e.g., any one of the exemplary Fc domains described herein) and the first domain in a pair of affinity domains (e.g., any one of the exemplary pair of affinity domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain in a pair of affinity domains (e.g., any of the exemplary pair of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are directly adjacent to each other in the third and fourth chimeric polypeptides. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the third and fourth chimeric polypeptides further comprise a linker sequence (e.g., any one of the exemplary linker sequences described herein or known in the art) between the second domain in the pair of affinity domains in the third and fourth chimeric polypeptides (e.g., any one of the exemplary pair of affinity domains described herein) and the second target binding domain (e.g., any one of the exemplary second target binding domains described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to different antigens. In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain (e.g., any of the exemplary second target binding domains described herein). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each an antigen binding domain (e.g., any of the exemplary second target binding domains described herein). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen binding domain (e.g., any of the exemplary second target binding domains described herein) comprises an scFv or a single domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any one of the exemplary target binding domains described herein or known in the art) specifically binds to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC A, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-. Beta.receptor II (TGF-. Beta.RII), ligands of DNAM1, ligands of NKp46, ligands of NKp44 a ligand for NKG2D, a ligand for NKp30, a ligand for scmhc i, a ligand for scmhc II, a ligand for scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD 28.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine protein. Non-limiting examples of soluble interleukin proteins and soluble cytokine proteins include: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine receptor. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include: soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3 or soluble CD28.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain can each independently specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands for TGF-beta receptor II (TGF-beta RII) a ligand for TGF-beta RIII, a ligand for DNAM1, a ligand for NKp46, a ligand for NKp44, a ligand for NKG2D, a ligand for NKp30, a ligand for scMHCI, a ligand for scTCR, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, and a receptor for CD 122.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein. In some embodiments of any one of the multi-chain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is selected from the group of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor or cytokine receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Tissue factor

Human tissue factor is a 263 amino acid transmembrane protein that contains three domains: (1) An N-terminal extracellular domain of 219 amino acids (residues 1-219); (2) A 22 amino acid transmembrane domain (residues 220-242); the cytoplasmic tail of (3) 21 amino acids (residues 242-263) ((UniProtKB identifier number: P13726): the cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258 and one S-palmitoylation site at Cys 245. No deletion or mutation of the cytoplasmic domain was found to affect tissue factor clotting activity. Tissue factor has one S-palmitoylation site at Cys245 in the intracellular domain of the protein. Cys245 is located at the amino acid terminus of the intracellular domain and near the membrane surface. Tissue factor transmembrane domain consists of a single span of a-helices.

The extracellular domain of tissue factor consisting of two fibronectin type III domains is linked to the transmembrane domain by a six amino acid linker. This linker provides conformational flexibility to decouple the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping beta sheets, with the top sheet domain containing three antiparallel beta strands and the bottom sheet containing four beta strands. Beta strands are linked between chains βa and βb, βc and βd, and βe and βf by a beta loop, which is conformationally conserved in both modules. There are three short alpha helical segments linked to the beta strand. A unique feature of tissue factor is the 17 amino acid β -hairpin between chain β10 and chain β11, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12 amino acid loop between β6F and β7G, which is absent from the C-terminal domain and is characteristic of tissue factor. This fibronectin type III domain structure is characteristic of immunoglobulin-like family protein folding and is conserved across a wide variety of extracellular proteins.

Once the zymogen FVII binds to tissue to form the active tissue factor-FVIIa complex, it can be rapidly converted to FVIIa by limited proteolysis. FVIIa circulates as an enzyme at a concentration of about 0.1nM (1% of plasma FVII) and can also bind directly to tissue factor. The allosteric interactions of tissue factor with FVIIa on the tissue factor-FVIIa complex greatly increase the enzymatic activity of FVIIa: the rate of hydrolysis of small chromogenic peptide substrates increases by about 20 to 100 fold, and the rate of activation of natural macromolecular substrates FIX and FX increases by nearly one million fold. Consistent with the allosteric activation of the FVIIa active site upon binding to tissue factor, formation of tissue factor-FVIIa complex on phospholipid bilayer (i.e. when phosphatidyl-L-serine is exposed at membrane surface) is inhibited with Ca ²⁺ The dependency increases FIX or FX activation rate by a factor of 1,000. About a million-fold overall increase in the tissue factor-FVIIa-phospholipid complex versus free FVIIa to FX activation is a key regulator of the coagulation cascade.

FVII is a single chain polypeptide of about 50kDa, consisting of 406 amino acid residues, with an N-terminal gamma carboxyglutamic acid (GLA) rich domain, two epidermal growth factor-like domains (EGF 1 and EFG 2) and a C-terminal serine protease domain. FVII is achieved by Ile- ¹⁵⁴ -Arg ¹⁵² Specific proteolytic cleavage of the bond is activated to FVIIa. This cleavage causes the light chainAnd heavy chain through Cys ¹³⁵ And Cys ²⁶² Are fixed together. FVIIa is expressed as Ca by its N-terminal GLA domain ²⁺ The dependent manner binds to the phospholipid membrane. The C-terminal end of the GLA domain is immediately adjacent to the aromatic stack and two EGF domains. Aromatic stacking connects GLA to bind a single Ca ²⁺ An ionic EGF1 domain. Occupying the Ca ²⁺ Binding sites increase FVIIa amide hydrolysis activity and tissue factor association. Catalytic triad is composed of His ¹⁹³ 、Asp ²⁴² And Ser ³⁴⁴ Composition, and single Ca within the FVIIa protease domain ²⁺ Binding of ions is critical to its catalytic activity. Proteolytic activation of FVII by FVIIa releases Ile ¹⁵³ At the newly formed amino terminus, folded back and inserted into an activation pocket, with Asp ³⁴³ Form salt bridges to create oxyanion pores. This salt bridge formation is critical for FVIIa activity. However, no oxyanion pore formation occurs in free FVIIa after proteolytic activation. As a result, FVIIa circulates in a zymogen-like state, which is not well recognized by plasma protease inhibitors, causing it to circulate with a half-life of about 90 minutes.

Tissue factor mediated localization of the FVIIa active site above the membrane surface is important for FVIIa towards cognate substrates. Free FVIIa adopts a stable extended structure when bound to the membrane, with its active site positioned about above the membrane surfaceAfter FVIIa binds to tissue factor, the FVa active site will be relocated to be closer to the membrane about +.>This modulation can help properly align the FVIIa catalytic triplet with the target substrate cleavage site. Using FVIIa without GLA domain, the active site has been shown to be located a similar distance above the membrane, demonstrating that tissue factor is able to fully support FVIIa active site localization even in the absence of FVIIa-membrane interactions. Additional data shows that as long as the tissue factor extracellular domain is tethered to the membrane surface in some way, the tissue Factors support full FVIIa proteolytic activity. However, the active site of FVIIa is increased above the membrane surface by more than +.>The ability of the tissue factor-FVIIa complex to activate FX is greatly reduced, but the amidolytic activity of tissue factor-FVIIa is not reduced.

Alanine scanning mutagenesis has been used to assess the effect of specific amino acid side chains in the extracellular domain of tissue factors on interactions with FVIIa (Gibbs et al, biochemistry 33 (47): 14003-14010, 1994; schullek et al, J Biol Chem 269 (30): 19399-19403, 1994). Alanine substitutions identify a limited number of residue positions at which the alanine substitution causes a 5 to 10 fold decrease in affinity for FVIIa binding. Most of these residue side chains were found to be sufficiently exposed to solvents in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand binding site is located over a broad region at the boundary between the two modules. In the C-module, residue Arg is located on the protruding B-C ring ¹³⁵ And Phe ¹⁴⁰ Providing independent contact with FVIIa. Leu (Leu) ¹³³ Is located at the base of the finger-like structure and is pressed into the slit between the two modules. This provides a solution consisting of Lys ²⁰ 、Thr ⁶⁰ 、Asp ⁵⁸ And Ile ²² Continuity of the main clusters of the constituent important binding residues. Thr (Thr) ⁶⁰ Only partially exposed to the solvent and may act as a local structure rather than being in significant contact with the ligand. The binding site extends to the concave surface of the intermodular corner, which involves Glu ²⁴ And Gln ¹¹⁰ And may involve more distant residues Val ²⁰⁷ . The binding region extends from Asp58 to Lys ⁴⁸ 、Lys ⁴⁶ 、Gln ³⁷ 、Asp ⁴⁴ And Trp ⁴⁵ And forming a convex surface region. Trp ⁴⁵ And Asp ⁴⁴ Does not independently interact with FVIIa, indicating Trp ⁴⁵ The effect of the mutation at the position may reflect that this side chain is adjacent to Asp ⁴⁴ And Gln ³⁷ The structural importance of the local stacking of side chains. The interaction region also comprises two surface-exposed aromatic residues Phe ⁷⁶ And Tyr ⁷⁸ Which is provided withA portion of the hydrophobic clusters are formed in the N-module.

Known physiological substrates for tissue factor-FVIIa are FVII, FIX and FX and certain protease activated receptors. Mutation analysis has identified a number of residues that, when mutated, support full FVIIa amide hydrolysis activity on small peptidyl substrates, but lack the ability to support activation of macromolecular substrates (i.e., FVII, FIX and FX) (Ruf et al, J Biol Chem267 (31): 22206-22210, 1992; ruf et al, J Biol Chem267 (9): 6375-6381, 1992; huang et al, J Biol Chem 271 (36): 21755-21757, 1996; kirchhofer et al, biochemistry 39 (25): 7380-7387, 2000). The tissue factor loop region at residues 159-165 and residues in or near this flexible loop have been shown to be critical for the proteolytic activity of the tissue factor-FVIIa complex. This defines the substrate binding site outer region of the proposed tissue factor, which is far from the FVIIa active site. Substitution of glycine residues with the slightly larger residue alanine greatly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility afforded by glycine is critical for the loop of residues 159-165 for recognition of tissue factor macromolecular substrates.

Also demonstrates the residue Lys ¹⁶⁵ And Lys ¹⁶⁶ Is important for substrate recognition and binding. Mutation of any of these residues to alanine causes a significant decrease in tissue factor cofactor function. In most tissue factor-FVIIa structures, lys ¹⁶⁵ And Lys ¹⁶⁶ Opposite to each other, lys ¹⁶⁵ Directed to FVIIa, lys ¹⁶⁶ Directed to the outer region of the substrate binding site in the crystal structure. Lys of FVIIa ¹⁶⁵ And Gla ³⁵ The putative salt bridge formation in between will support the notion that the GLA domain interaction of tissue factor with FVIIa regulates substrate recognition. These results indicate that the C-terminal part of the extracellular domain of tissue factor interacts directly with the GLA domains of FIX and FX, possibly the adjacent EGF1 domain, and that the presence of FVIIa GLA domain can directly or indirectly modulate these interactions.

Soluble tissue factor domains

In some embodiments of one of the polypeptides, compositions or methods described herein, the soluble tissue factor domain may be a wild-type tissue factor polypeptide lacking a signal sequence, a transmembrane domain and an intracellular domain. In some embodiments, the soluble tissue factor domain may be a tissue factor mutant, wherein the wild-type tissue factor polypeptide lacks a signal sequence, a transmembrane domain, and an intracellular domain, and has been further modified at a selected amino acid. In some embodiments, the soluble tissue factor domain may be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain may be a rat soluble tissue factor domain. Non-limiting examples of soluble human tissue factor domains, mouse soluble tissue factor domains, rat soluble tissue factor domains, and mutant soluble tissue factor domains are shown below.

Exemplary soluble human tissue factor Domain (SEQ ID NO: 93)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

Exemplary nucleic acids encoding soluble human tissue factor domains (SEQ ID NO: 94)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

Exemplary soluble mouse tissue factor Domain (SEQ ID NO: 95)

agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge

Exemplary soluble rat tissue factor Domain (SEQ ID NO: 96)

Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtkigqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

Exemplary mutant soluble human tissue factor Domain (SEQ ID NO: 97)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

Exemplary mutant soluble human tissue factor Domain (SEQ ID NO: 98)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

In some embodiments, the soluble tissue factor domain may comprise a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID No. 93, 95, 96, 97, or 98. In some embodiments, the soluble tissue factor domain may comprise the sequence of SEQ ID NO 93, 95, 96, 97, or 98, with one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) removed at the N-terminus and/or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) removed at the C-terminus.

As will be appreciated by those skilled in the art, mutations of amino acids that are conserved between different mammalian species are more likely to reduce the activity and/or structural stability of the protein, while mutations of amino acids that are not conserved between different mammalian species are less likely to reduce the activity and/or structural stability of the protein.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain is not capable of binding to factor VIIa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any one of the multi-chain chimeric polypeptides described herein, the multi-chain chimeric polypeptide does not stimulate coagulation in a mammal.

In some embodiments, the soluble tissue factor domain may be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble rat tissue factor domain.

In some embodiments, the soluble tissue factor domain does not include one or more (e.g., two, three, four, five, six, or seven) of: lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein; arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the mutant soluble tissue factor has the amino acid sequence of SEQ ID NO. 97 or SEQ ID NO. 98.

In some embodiments, the soluble tissue factor domain may be encoded by a nucleic acid comprising a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID No. 94.

In some embodiments of the present invention, in some embodiments, the total length of the soluble tissue factor domain can be from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 160 amino acids, from about 20 amino acids to about 155 amino acids, from about 20 amino acids to about 150 amino acids, from about 20 amino acids to about 145 amino acids, from about 20 amino acids to about 140 amino acids about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 40 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 215 amino acids, about 40 amino acids to about 210 amino acids, about 40 amino acids to about 205 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 215 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 205 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 215 amino acids, about 60 amino acids to about 210 amino acids, about 60 amino acids to about 205 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids about 60 amino acids to about 70 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids, about 130 amino acids to about 210 amino acids, about 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids about 135 amino acids to about 140 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 140 amino acids, about 145 to about 215 amino acids, about 145 to about 210 amino acids, about 145 to about 205 amino acids, about 145 to about 200 amino acids, about 145 to about 195 amino acids, about 145 to about 190 amino acids, about 145 to about 185 amino acids, about 145 to about 180 amino acids, about 145 to about 175 amino acids, about 145 to about 170 amino acids, about 145 to about 165 amino acids, about 145 to about 160 amino acids, about 145 to about 155 amino acids, about 145 to about 150 amino acids, about 150 to about 220 amino acids, about 150 to about 215 amino acids, about 150 to about 210 amino acids, about 150 to about 205 amino acids, about 150 to about 200 amino acids about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids, about, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids about 165 to about 205 amino acids, about 165 to about 200 amino acids, about 165 to about 195 amino acids, about 165 to about 190 amino acids, about 165 to about 185 amino acids, about 165 to about 180 amino acids, about 165 to about 175 amino acids, about 165 to about 170 amino acids, about 170 to about 220 amino acids, about 170 to about 215 amino acids, about 170 to about 210 amino acids, about 170 to about 205 amino acids, about 170 to about 200 amino acids, about 170 to about 195 amino acids, about 170 to about 190 amino acids, about 170 to about 185 amino acids, about 170 to about 180 amino acids, about 170 to about 175 amino acids, about 175 to about 220 amino acids, about 175 to about 215 amino acids, about 175 to about 210 amino acids, about 175 to about 205 amino acids, about 175 to about 200 amino acids, about 175 to about 195 amino acids, about 175 to about 190 amino acids, about 175 to about 185 amino acids, about 175 to about 180 amino acids, about 180 to about 220 amino acids, about 180 to about 215 amino acids, about 180 to about 210 amino acids, about 180 to about 205 amino acids, about 180 to about 200 amino acids, about 180 to about 195 amino acids, about 180 to about 190 amino acids, about 185 to about 185 amino acids, about 185 to about 215 amino acids about 185 amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195 amino acids to about 205 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215 amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205 amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids, about 210 amino acids to about 220 amino acids, about 210 amino acids to about 215 amino acids, or about 215 amino acids to about 220 amino acids.

Linker sequences

In some embodiments, the linker sequence may be a flexible linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et al, protein Engineering, design & Selection 27 (10): 325-330, 2014; priyanka et al, protein sci.22 (2): 153-167, 2013. In some embodiments, the linker sequence is a synthetic linker sequence.

In some embodiments, any of the single-chain chimeric polypeptides described herein can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments, any of the single-chain chimeric polypeptides described herein can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

In some embodiments of the present invention, in some embodiments, the total length of the linker sequence may be from 1 amino acid to about 100 amino acids, from 1 amino acid to about 90 amino acids, from 1 amino acid to about 80 amino acids, from 1 amino acid to about 70 amino acids, from 1 amino acid to about 60 amino acids, from 1 amino acid to about 50 amino acids, from 1 amino acid to about 45 amino acids, from 1 amino acid to about 40 amino acids, from 1 amino acid to about 35 amino acids, from 1 amino acid to about 30 amino acids, from 1 amino acid to about 25 amino acids, from 1 amino acid to about 24 amino acids, from 1 amino acid to about 22 amino acids, from 1 amino acid to about 20 amino acids, from 1 amino acid to about 18 amino acids, from 1 amino acid to about 16 amino acids, from 1 amino acid to about 14 amino acids, from 1 amino acid to about 12 amino acids, from 1 amino acid to about 10 amino acids, from 1 amino acid to about 1 amino acid to about 8 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 4 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 100 amino acids, about 12 amino acids to about 90 amino acids, about 12 amino acids to about 80 amino acids, about 12 amino acids to about 70 amino acids, about 12 amino acids to about 60 amino acids, about 12 amino acids to about 50 amino acids, about 12 amino acids to about 45 amino acids, about 12 amino acids to about 40 amino acids about 12 amino acids to about 35 amino acids, about 12 amino acids to about 30 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14 amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids, about 14 amino acids to about 60 amino acids, about 14 amino acids to about 50 amino acids, about 14 amino acids to about 45 amino acids, about 14 amino acids to about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids, about, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, about 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 100 amino acids, about 22 amino acids to about 90 amino acids, about 22 amino acids to about 80 amino acids, about 22 amino acids to about 70 amino acids, about 22 amino acids to about 60 amino acids, about 22 amino acids to about 50 amino acids, about 22 amino acids to about 45 amino acids about 22 amino acids to about 40 amino acids, about 22 amino acids to about 35 amino acids, about 22 amino acids to about 30 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino acids, about, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 90 amino acids, or about 90 amino acids to about 100 amino acids.

In some embodiments, the linker is enriched in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is enriched in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 99) sequences, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGGS (SEQ ID NO: 99) sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGGGGGS (SEQ ID NO: 100) sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 101) sequences, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGSG (SEQ ID NO: 101) sequences. In some embodiments, the linker comprises GGSSRSSSSGGGGSGGGG (SEQ ID NO: 222).

In some embodiments, the linker sequence may comprise or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 102). In some embodiments, the linker sequence may be encoded by a nucleic acid comprising or consisting of: GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 103). In some embodiments, the linker sequence may comprise or consist of: GGGSGGGS (SEQ ID NO: 104).

Target binding domain

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and/or the additional one or more target binding domains can be an antigen binding domain (e.g., any of the exemplary antigen binding domains described herein or known in the art), a soluble interleukin or cytokine protein (e.g., any of the exemplary soluble interleukin proteins or soluble cytokine proteins described herein), and a soluble interleukin or cytokine receptor (e.g., any of the exemplary soluble interleukin receptors or soluble cytokine receptors described herein).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and/or one or more additional target binding domains may each independently have a total of about 5 amino acids to about 1000 amino acids, about 5 amino acids to about 950 amino acids, about 5 amino acids to about 900 amino acids, about 5 amino acids to about 850 amino acids, about 5 amino acids to about 800 amino acids, about 5 amino acids to about 750 amino acids, about 5 amino acids to about 700 amino acids, about 5 amino acids to about 650 amino acids, about 5 amino acids to about 600 amino acids, about 5 amino acids to about 550 amino acids, about 5 amino acids to about 500 amino acids, about 5 amino acids to about 450 amino acids, about 5 amino acids to about 400 amino acids, about 5 amino acids to about 350 amino acids, about 5 amino acids to about 300 amino acids, about 5 amino acids to about 280 amino acids, about 5 amino acids to about 750 amino acids, about 5 amino acids to about 260 amino acids to about 150 amino acids, about 180 amino acids to about 180 amino acids, about 5 amino acids to about 180 amino acids, about 150 amino acids to about 190 amino acids, about 5 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 5 amino acids to about 150 amino acids, about 150 amino acids to about 150 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115 amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about 105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about45 amino acids about 5 amino acids to about 40 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids, about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids about 10 amino acids to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 55 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 1000 amino acids, about 15 amino acids to about 950 amino acids, about 15 amino acids to about 900 amino acids, about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700 amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to about 600 amino acids about 15 amino acids to about 550 amino acids, about 15 amino acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15 amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids, about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino acids, about 15 amino acids to about 260 amino acids, about 15 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids to about 125 amino acids, about 15 amino acids to about 120 amino acids, about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 95 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids about 15 amino acids to about 65 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 1000 amino acids, about 20 amino acids to about 950 amino acids, about 20 amino acids to about 900 amino acids, about 20 amino acids to about 850 amino acids, about 20 amino acids to about 800 amino acids, about 20 amino acids to about 750 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 1000 amino acids, about 25 amino acids to about 950 amino acids, about 25 amino acids to about 900 amino acids, about 25 amino acids to about 850 amino acids, about 25 amino acids to about 800 amino acids, about 25 amino acids to about 750 amino acids, about 25 amino acids to about 700 amino acids, about 25 amino acids to about 650 amino acids, about 25 amino acids to about 600 amino acids about 25 amino acids to about 550 amino acids, about 25 amino acids to about 500 amino acids, about 25 amino acids to about 450 amino acids, about 25 amino acids to about 400 amino acids, about 25 amino acids to about 350 amino acids, about 25 amino acids to about 300 amino acids, about 25 amino acids to about 280 amino acids, about 25 amino acids to about 260 amino acids, about 25 amino acids to about 240 amino acids, about 25 amino acids to about 220 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino acids to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25 amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 25 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 1000 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 65 amino acids, about, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 55 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 1000 amino acids, about 35 amino acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35 amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids, about 35 amino acids to about 750 amino acids, about 35 amino acids to about 700 amino acids, about 35 amino acids to about 650 amino acids, about 35 amino acids to about 600 amino acids, about 35 amino acids to about 550 amino acids, about 35 amino acids to about 500 amino acids, about 35 amino acids to about 450 amino acids, about 35 amino acids to about 400 amino acids about 35 amino acids to about 350 amino acids, about 35 amino acids to about 300 amino acids, about 35 amino acids to about 280 amino acids, about 35 amino acids to about 260 amino acids, about 35 amino acids to about 240 amino acids, about 35 amino acids to about 220 amino acids, about 35 amino acids to about 200 amino acids, about 35 amino acids to about 195 amino acids, about 35 amino acids to about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino acids to about 180 amino acids, about 35 amino acids to about 175 amino acids, about 35 amino acids to about 170 amino acids, about 35 amino acids to about 165 amino acids, about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino acids, about 35 amino acids to about 150 amino acids, about 35 amino acids to about 145 amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 125 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids about 35 amino acids to about45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 1000 amino acids, about 40 amino acids to about 950 amino acids, about 40 amino acids to about 900 amino acids, about 40 amino acids to about 850 amino acids, about 40 amino acids to about 800 amino acids, about 40 amino acids to about 750 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40 amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about45 amino acids, about45 amino acids to about 1000 amino acids, about45 amino acids to about 950 amino acids, about45 amino acids to about 900 amino acids, about45 amino acids to about 850 amino acids, about45 amino acids to about 800 amino acids, about45 amino acids to about 750 amino acids, about45 amino acids to about 700 amino acids, about45 amino acids to about 650 amino acids, about45 amino acids to about 600 amino acids, about45 amino acids to about 550 amino acids, about45 amino acids to about 500 amino acids, about45 amino acids to about 450 amino acids, about45 amino acids to about 400 amino acids, about45 amino acids to about 350 amino acids, about45 amino acids to about 300 amino acids, about45 amino acids to about 280 amino acids, about45 amino acids to about 260 amino acids, about45 amino acids to about 240 amino acids, about45 amino acids to about 220 amino acids, about45 amino acids to about 200 amino acids, about45 amino acids to about 195 amino acids about45 amino acids to about 190 amino acids, about45 amino acids to about 185 amino acids, about45 amino acids to about 180 amino acids, about45 amino acids to about 175 amino acids, about45 amino acids to about 170 amino acids, about45 amino acids to about 165 amino acids, about45 amino acids to about 160 amino acids, about45 amino acids to about 155 amino acids, about45 amino acids to about 150 amino acids, about45 amino acids to about 145 amino acids, about45 amino acids to about 140 amino acids, about45 amino acids to about 135 amino acids, about45 amino acids to about 130 amino acids, about45 amino acids to about 125 amino acids, about45 amino acids to about 120 amino acids, about45 amino acids to about 115 amino acids, about45 amino acids to about 110 amino acids, about45 amino acids to about 105 amino acids, about45 amino acids to about 100 amino acids, about45 amino acids to about 95 amino acids, about45 amino acids to about 90 amino acids, about45 amino acids to about 85 amino acids, about45 amino acids to about 80 amino acids, about45 amino acids to about 75 amino acids, about45 amino acids to about 70 amino acids, about45 amino acids to about 65 amino acids, about45 amino acids to about 60 amino acids, about45 amino acids to about 55 amino acids, about45 amino acids to about 50 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids o amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids, about 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids, about 400 amino acids, about 55 amino acids to about 350 amino acids, about 55 amino acids to about 300 amino acids, about 55 amino acids to about 280 amino acids, about 55 amino acids to about 260 amino acids, about 55 amino acids to about 240 amino acids, about 55 amino acids to about 220 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60 amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 65 amino acids to about 65 amino acids, about 65 amino acids to about 1000 amino acids, about 65 amino acids to about 950 amino acids, about 65 amino acids to about 900 amino acids, about 65 amino acids to about 850 amino acids, about 65 amino acids to about 800 amino acids, about 65 amino acids to about 750 amino acids, about 65 amino acids to about 700 amino acids, about 65 amino acids to about 650 amino acids, about 65 amino acids to about 600 amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to about 500 amino acids, about 65 amino acids to about 450 amino acids about 65 amino acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65 amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids, about 65 amino acids to about 260 amino acids, about 65 amino acids to about 240 amino acids, about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 950 amino acids, about 70 amino acids to about 900 amino acids about 70 amino acids to about 850 amino acids, about 70 amino acids to about 800 amino acids, about 70 amino acids to about 750 amino acids, about 70 amino acids to about 700 amino acids, about 70 amino acids to about 650 amino acids, about 70 amino acids to about 600 amino acids, about 70 amino acids to about 550 amino acids, about 70 amino acids to about 500 amino acids, about 70 amino acids to about 450 amino acids, about 70 amino acids to about 400 amino acids, about 70 amino acids to about 350 amino acids, about 70 amino acids to about 300 amino acids, about 70 amino acids to about 280 amino acids, about 70 amino acids to about 260 amino acids, about 70 amino acids to about 240 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 1000 amino acids, about 75 amino acids to about 950 amino acids, about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino acids, about 75 amino acids to about 800 amino acids, about 75 amino acids to about 750 amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75 amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids, about 75 amino acids to about 350 amino acids, about 75 amino acids to about 300 amino acids, about 75 amino acids to about 280 amino acids, about 75 amino acids to about 260 amino acids, about 75 amino acids to about 240 amino acids, about 75 amino acids to about 220 amino acids, about 75 amino acids to about 200 amino acids, about 75 amino acids to about 195 amino acids, about 75 amino acids to about 190 amino acids, about 75 amino acids to about 185 amino acids, about 75 amino acids to about 180 amino acids, about 75 amino acids to about 175 amino acids, about 75 amino acids to about 170 amino acids, about 75 amino acids to about 165 amino acids, about 75 amino acids to about 160 amino acids, about 75 amino acids to about 155 amino acids, about 75 amino acids to about 150 amino acids, about 75 amino acids to about 145 amino acids, about 75 amino acids to about 140 amino acids about 75 amino acids to about 135 amino acids, about 75 amino acids to about 130 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 1000 amino acids, about 80 amino acids to about 950 amino acids, about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino acids, about 80 amino acids to about 800 amino acids, about 80 amino acids to about 750 amino acids, about 75 amino acids to about 85 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids, about 80 amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids, about 80 amino acids to about 350 amino acids, about 80 amino acids to about 300 amino acids, about 80 amino acids to about 280 amino acids, about 80 amino acids to about 260 amino acids, about 80 amino acids to about 240 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 80 amino acids, about 85 amino acids to about 1000 amino acids, about 85 amino acids to about 950 amino acids, about 85 amino acids to about 900 amino acids, about 85 amino acids to about 850 amino acids, about 85 amino acids to about 800 amino acids, about 85 amino acids to about 750 amino acids, about 85 amino acids to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 260 amino acids, about 85 amino acids to about 240 amino acids, about 85 amino acids to about 220 amino acids about 85 amino acids to about 200 amino acids, about 85 amino acids to about 195 amino acids, about 85 amino acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85 amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155 amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to about 145 amino acids, about 85 amino acids to about 140 amino acids, about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85 amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids, about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90 amino acids to about 850 amino acids, about 90 amino acids to about 800 amino acids, about 90 amino acids to about 750 amino acids, about 90 amino acids to about 700 amino acids, about 90 amino acids to about 650 amino acids, about 90 amino acids to about 600 amino acids, about 90 amino acids to about 550 amino acids, about 90 amino acids to about 500 amino acids, about 90 amino acids to about 450 amino acids, about 90 amino acids to about 400 amino acids, about 90 amino acids to about 350 amino acids about 90 amino acids to about 300 amino acids, about 90 amino acids to about 280 amino acids, about 90 amino acids to about 260 amino acids, about 90 amino acids to about 240 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 1000 amino acids, about 95 amino acids to about 950 amino acids, about 95 amino acids to about 900 amino acids, about 95 amino acids to about 850 amino acids, about 95 amino acids to about 800 amino acids, about 95 amino acids to about 750 amino acids, about 95 amino acids to about 700 amino acids, about 95 amino acids to about 650 amino acids, about 95 amino acids to about 600 amino acids, about 95 amino acids to about 550 amino acids about 95 amino acids to about 500 amino acids, about 95 amino acids to about 450 amino acids, about 95 amino acids to about 400 amino acids, about 95 amino acids to about 350 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 280 amino acids, about 95 amino acids to about 260 amino acids, about 95 amino acids to about 240 amino acids, about 95 amino acids to about 220 amino acids, about 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 to about 155 amino acids, about 95 to about 150 amino acids, about 95 to about 145 amino acids, about 95 to about 140 amino acids, about 95 to about 135 amino acids, about 95 to about 130 amino acids, about 95 to about 125 amino acids, about 95 to about 120 amino acids, about 95 to about 115 amino acids, about 95 to about 110 amino acids, about 95 to about 105 amino acids, about 95 to about 100 amino acids, about 100 to about 1000 amino acids, about 100 to about 950 amino acids, about 100 to about 900 amino acids, about 100 to about 850 amino acids, about 100 to about 800 amino acids, about 100 to about 750 amino acids, about 100 to about 700 amino acids about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 1000 amino acids, about 105 amino acids to about 950 amino acids, about 105 amino acids to about 900 amino acids, about 105 amino acids to about 850 amino acids, about 105 amino acids to about 800 amino acids about 105 amino acids to about 750 amino acids, about 105 amino acids to about 700 amino acids, about 105 amino acids to about 650 amino acids, about 105 amino acids to about 600 amino acids, about 105 amino acids to about 550 amino acids, about 105 amino acids to about 500 amino acids, about 105 amino acids to about 450 amino acids, about 105 amino acids to about 400 amino acids, about 105 amino acids to about 350 amino acids, about 105 amino acids to about 300 amino acids, about 105 amino acids to about 280 amino acids, about 105 amino acids to about 260 amino acids, about 105 amino acids to about 240 amino acids, about 105 amino acids to about 220 amino acids, about 105 amino acids to about 200 amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 to about 180 amino acids, about 105 to about 175 amino acids, about 105 to about 170 amino acids, about 105 to about 165 amino acids, about 105 to about 160 amino acids, about 105 to about 155 amino acids, about 105 to about 150 amino acids, about 105 to about 145 amino acids, about 105 to about 140 amino acids, about 105 to about 135 amino acids, about 105 to about 130 amino acids, about 105 to about 125 amino acids, about 105 to about 120 amino acids, about 105 to about 115 amino acids, about 105 to about 110 amino acids, about 110 to about 1000 amino acids, about 110 to about 950 amino acids, about 110 to about 850 amino acids, and a process for preparing a pharmaceutical composition about 110 amino acids to about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110 amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids, about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550 amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to about 450 amino acids, about 110 amino acids to about 400 amino acids, about 110 amino acids to about 350 amino acids, about 110 amino acids to about 300 amino acids, about 110 amino acids to about 280 amino acids, about 110 amino acids to about 260 amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids about 115 amino acids to about 800 amino acids, about 115 amino acids to about 750 amino acids, about 115 amino acids to about 700 amino acids, about 115 amino acids to about 650 amino acids, about 115 amino acids to about 600 amino acids, about 115 amino acids to about 550 amino acids, about 115 amino acids to about 500 amino acids, about 115 amino acids to about 450 amino acids, about 115 amino acids to about 400 amino acids, about 115 amino acids to about 350 amino acids, about 115 amino acids to about 300 amino acids, about 115 amino acids to about 280 amino acids, about 115 amino acids to about 260 amino acids, about 115 amino acids to about 240 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to about 600 amino acids, about 120 amino acids to about 550 amino acids, about 120 amino acids to about 500 amino acids, about 120 amino acids to about 450 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350 amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids to about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120 amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 1000 amino acids, about 125 amino acids to about 950 amino acids, about 125 amino acids to about 900 amino acids, about 125 amino acids to about 850 amino acids, about 125 amino acids to about 800 amino acids, about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700 amino acids about 125 amino acids to about 650 amino acids, about 125 amino acids to about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125 amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350 amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to about 280 amino acids, about 125 amino acids to about 260 amino acids, about 125 amino acids to about 240 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 1000 amino acids, about 130 amino acids to about 950 amino acids, about 130 amino acids to about 900 amino acids, about 130 amino acids to about 850 amino acids, about 130 amino acids to about 800 amino acids, about 130 amino acids to about 750 amino acids, about 130 amino acids to about 700 amino acids, about 130 amino acids to about 650 amino acids, about 130 amino acids to about 600 amino acids, about 130 amino acids to about 550 amino acids about 130 amino acids to about 500 amino acids, about 130 amino acids to about 450 amino acids, about 130 amino acids to about 400 amino acids, about 130 amino acids to about 350 amino acids, about 130 amino acids to about 300 amino acids, about 130 amino acids to about 280 amino acids, about 130 amino acids to about 260 amino acids, about 130 amino acids to about 240 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids, about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900 amino acids, about 135 amino acids to about 850 amino acids, about 135 amino acids to about 800 amino acids, about 135 amino acids to about 750 amino acids, about 135 amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids, about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550 amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to about 450 amino acids, about 135 amino acids to about 400 amino acids, about 135 amino acids to about 350 amino acids about 135 amino acids to about 300 amino acids, about 135 amino acids to about 280 amino acids, about 135 amino acids to about 260 amino acids, about 135 amino acids to about 240 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 135 amino acids to about 150 amino acids, about 140 amino acids to about 1000 amino acids, about 140 amino acids to about 950 amino acids, about 140 amino acids to about 900 amino acids, about 140 amino acids to about 850 amino acids, about 140 amino acids to about 800 amino acids, about 140 amino acids to about 750 amino acids, about 140 amino acids to about 700 amino acids, about 140 amino acids to about 650 amino acids, about 140 amino acids to about 600 amino acids, about 140 amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 350 amino acids, about 140 amino acids to about 300 amino acids, about 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids, about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900 amino acids, about 145 amino acids to about 850 amino acids, about 145 amino acids to about 800 amino acids, about 145 amino acids to about 750 amino acids, about 140 amino acids to about 1000 amino acids, about 145 to about 700 amino acids, about 145 to about 650 amino acids, about 145 to about 600 amino acids, about 145 to about 550 amino acids, about 145 to about 500 amino acids, about 145 to about 450 amino acids, about 145 to about 400 amino acids, about 145 to about 350 amino acids, about 145 to about 300 amino acids, about 145 to about 280 amino acids, about 145 to about 260 amino acids, about 145 to about 240 amino acids, about 145 to about 220 amino acids, about 145 to about 200 amino acids, about 145 to about 195 amino acids, about 145 to about 190 amino acids, about 145 to about 185 amino acids, about 180 amino acids, about 145 to about 175 amino acids about 145 to about 170 amino acids, about 145 to about 165 amino acids, about 145 to about 160 amino acids, about 145 to about 155 amino acids, about 145 to about 150 amino acids, about 150 to about 1000 amino acids, about 150 to about 950 amino acids, about 150 to about 900 amino acids, about 150 to about 850 amino acids, about 150 to about 800 amino acids, about 150 to about 750 amino acids, about 150 to about 700 amino acids, about 150 to about 650 amino acids, about 150 to about 600 amino acids, about 150 to about 550 amino acids, about 150 to about 500 amino acids, about 150 to about 450 amino acids, about 150 to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 1000 amino acids, about 155 amino acids to about 950 amino acids, about 155 amino acids to about 900 amino acids about 155 amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids, about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700 amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155 amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids, about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350 amino acids, about 155 amino acids to about 300 amino acids, about 155 amino acids to about 280 amino acids, about 155 amino acids to about 260 amino acids, about 155 amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to about 950 amino acids, about 160 amino acids to about 900 amino acids, about 160 amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids, about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700 amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350 amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 1000 amino acids, about 165 amino acids to about 950 amino acids, about 165 amino acids to about 900 amino acids, about 165 amino acids about 850 amino acids, about 165 amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids, about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650 amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165 amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids, about 165 amino acids to about 350 amino acids, about 165 amino acids to about 300 amino acids, about 165 amino acids to about 280 amino acids, about 165 amino acids to about 260 amino acids, about 165 amino acids to about 240 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids about 165 to about 185 amino acids, about 165 to about 180 amino acids, about 165 to about 175 amino acids, about 165 to about 170 amino acids, about 170 to about 1000 amino acids, about 170 to about 950 amino acids, about 170 to about 900 amino acids, about 170 to about 850 amino acids, about 170 to about 800 amino acids, about 170 to about 750 amino acids, about 170 to about 700 amino acids, about 170 to about 650 amino acids, about 170 to about 600 amino acids, about 170 to about 550 amino acids, about 170 to about 500 amino acids, about 170 to about 450 amino acids, about 170 to about 400 amino acids, about 170 to about 350 amino acids, about 170 amino acids to about 300 amino acids, about 170 amino acids to about 280 amino acids, about 170 amino acids to about 260 amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175 amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids, about 175 amino acids to about 800 amino acids, about 175 amino acids to about 750 amino acids, about 175 amino acids to about 700 amino acids, about 175 amino acids to about 650 amino acids about 175 amino acids to about 600 amino acids, about 175 amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids, about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175 amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids, about 180 amino acids to about 950 amino acids, about 180 amino acids to about 900 amino acids, about 180 amino acids to about 850 amino acids, about 180 amino acids to about 800 amino acids about 180 amino acids to about 750 amino acids, about 180 amino acids to about 700 amino acids, about 180 amino acids to about 650 amino acids, about 180 amino acids to about 600 amino acids, about 180 amino acids to about 550 amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to about 450 amino acids, about 180 amino acids to about 400 amino acids, about 180 amino acids to about 350 amino acids, about 180 amino acids to about 300 amino acids, about 180 amino acids to about 280 amino acids, about 180 amino acids to about 260 amino acids, about 180 amino acids to about 240 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 1000 amino acids, about 185 amino acids to about 950 amino acids, about 185 amino acids to about 185 amino acids 900 amino acids, about 185 amino acids to about 850 amino acids, about 185 amino acids to about 800 amino acids, about 185 amino acids to about 750 amino acids, about 185 amino acids to about 700 amino acids, about 185 amino acids to about 650 amino acids, about 185 amino acids to about 600 amino acids, about 185 amino acids to about 550 amino acids, about 185 amino acids to about 500 amino acids, about 185 amino acids to about 450 amino acids, about 185 amino acids to about 400 amino acids, about 185 amino acids to about 350 amino acids, about 185 amino acids to about 300 amino acids, about 185 amino acids to about 280 amino acids, about 185 amino acids to about 260 amino acids, about 185 amino acids to about 240 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 1000 amino acids, about 190 amino acids to about 950 amino acids, about 190 amino acids to about 900 amino acids, about 190 amino acids to about 850 amino acids, about 190 amino acids to about 800 amino acids, about 190 amino acids to about 750 amino acids, about 190 amino acids to about 700 amino acids, about 190 amino acids to about 650 amino acids, about 190 amino acids to about 600 amino acids, about 190 amino acids to about 550 amino acids, about 190 amino acids to about 500 amino acids, about 190 amino acids to about 450 amino acids, about 190 amino acids to about 400 amino acids about 190 amino acids to about 350 amino acids, about 190 amino acids to about 300 amino acids, about 190 amino acids to about 280 amino acids, about 190 amino acids to about 260 amino acids, about 190 amino acids to about 240 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 1000 amino acids, about 195 amino acids to about 950 amino acids, about 195 amino acids to about 900 amino acids, about 195 amino acids to about 850 amino acids, about 195 amino acids to about 800 amino acids, about 195 amino acids to about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids, about 195 to about 500 amino acids, about 195 to about 450 amino acids, about 195 to about 400 amino acids, about 195 to about 350 amino acids, about 195 to about 300 amino acids, about 195 to about 280 amino acids, about 195 to about 260 amino acids, about 195 to about 240 amino acids, about 195 to about 220 amino acids, about 195 to about 200 amino acids, about 200 to about 1000 amino acids, about 200 to about 950 amino acids, about 200 to about 900 amino acids, about 200 to about 850 amino acids, about 200 to about 800 amino acids, about 200 to about 750 amino acids, about 200 to about 700 amino acids, about 200 to about 650 amino acids, about 200 to about 600 amino acids about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids, about, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 450 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 350 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 450 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 350 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 450 amino acids, about 280 amino acids to about 400 amino acids about 280 amino acids to about 350 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 1000 amino acids, about 350 amino acids to about 950 amino acids, about 300 amino acids, about 350 amino acids to about 900 amino acids, about 350 amino acids to about 850 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 1000 amino acids, about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids about 600 amino acids about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, or about 950 amino acids to about 1000 amino acids.

Any of the target binding domains described herein can be less than 1x10 ^-7 M is less than 1x10 ^-8 M is less than 1x10 ^-9 M is less than 1x10 ^-10 M is less than 1x10 ^-11 M is less than 1x10 ^-12 M or less than 1x10 ^-13 Dissociation equilibrium constant of M (K _D ) To bind to its target. In some embodiments, the antigen binding protein constructs provided herein can be at about 1x10 ^-3 M to about 1x10 ^-5 M, about 1x10 ^-4 M to about 1x10 ^-6 M, about 1x10 ^-5 M to about 1x10 ^-7 M, about 1x10 ^-6 M to about 1x10 ^-8 M, about 1x10 ^-7 M to about 1x10 ^-9 M, about 1x10 ^-8 M to about 1x10 ^-10 M or about 1x10 ^-9 M to about 1x10 ^-11 M (inclusive) K _D Binds to the recognition antigen.

Any of the target binding domains described herein can be present at between about 1pM to about 30nM (e.g., about 1pM to about 25nM, about 1pM to about 20nM, about 1pM to about 15nM, about 1pM to about 10nM, about 1pM to about 5nM, about 1pM to about 2nM, about 1pM to about 1nM, about 1pM to about 950pM, about 1pM to about 900pM, about 1pM to about 850pM, about 1pM to about 800pM, about 1pM to about 750pM, about 1pM to about 700pM, about 1pM to about 650pM, about 1pM to about 600pM, about 1pM to about 550pM, about 1pM to about 500pM, about 1pM to about 450pM, about 1pM to about 400pM, about 1pM to about 350pM about 1pM to about 300pM, about 1pM to about 250pM, about 1pM to about 200pM, about 1pM to about 150pM, about 1pM to about 100pM, about 1pM to about 90pM, about 1pM to about 80pM, about 1pM to about 70pM, about 1pM to about 60pM, about 1pM to about 50pM, about 1pM to about 40pM, about 1pM to about 30pM, about 1pM to about 20pM, about 1pM to about 10pM, about 1pM to about 5pM, about 1pM to about 4pM, about 1pM to about 3pM, about 1pM to about 2pM, about 2pM to about 30nM, about 2pM to about 25nM, about 2pM to about 20nM about 1pM to about 300pM, about 1pM to about 250pM, about 1pM to about 200pM, about 1pM to about 150pM, about 1pM to about 100pM, about 1pM to about 90pM, about 1pM to about 80pM, about 1pM to about 70pM, about 1pM to about 60pM, about 1pM to about 50pM, a method of manufacturing the same, and a method of manufacturing the same about 1pM to about 40pM, about 1pM to about 30pM, about 1pM to about 20pM, about 1pM to about 10pM, about 1pM to about 5pM, about 1pM to about 4pM, about 1pM to about 3pM, about 1pM to about 2pM, about 2pM to about 30nM, about 2pM to about 25nM, about 2pM to about 20nM, about 5pM to about 5nM, about 5pM to about 2nM, about 5pM to about 1nM, about 5pM to about 950pM, about 5pM to about 900pM, about 5pM to about 850pM, about 5pM to about 800pM, about 5pM to about 750pM, about 5pM to about 700pM, about 5pM to about 650pM, about 5pM to about 600pM, about 5pM to about 550pM, about 5pM to about 500pM, about 5pM to about 450pM, about 5pM to about 400pM, about 5pM to about 350pM, about 5pM to about 300pM, about 5pM to about 250pM, about 5pM to about 200pM, about 5pM to about 150pM, about 5pM to about 100pM, about 5pM to about 90pM, about 5pM to about 80pM, about 5pM to about 70pM, about 5pM to about 60pM, about 5pM to about 50pM, about 5pM to about 40pM about 5pM to about 30pM, about 5pM to about 20pM, about 5pM to about 10pM, about 10pM to about 30nM, about 10pM to about 25nM, about 10pM to about 20nM, about 10pM to about 15nM, about 10pM to about 10nM, about 10pM to about 5nM, about 10pM to about 2nM, about 10pM to about 1nM, about 10pM to about 950pM, about 10pM to about 900pM, about 10pM to about 850pM, about 10pM to about 800pM, about 10pM to about 750pM, about 10pM to about 700pM, about 10pM to about 650pM, about 10pM to about 600pM, about 10pM to about 550pM, about 10pM to about 500pM about 10pM to about 450pM, about 10pM to about 400pM, about 10pM to about 350pM, about 10pM to about 300pM, about 10pM to about 250pM, about 10pM to about 200pM, about 10pM to about 150pM, about 10pM to about 100pM, about 10pM to about 90pM, about 10pM to about 80pM, about 10pM to about 70pM, about 10pM to about 60pM, about 10pM to about 50pM, about 10pM to about 40pM, about 10pM to about 30pM, about 10pM to about 20pM, about 15pM to about 30nM, about 15pM to about 25nM, about 15pM to about 20nM, about 15pM to about 15nM, about 15pM to about 10nM, about 15pM to about 5nM about 15pM to about 2nM, about 15pM to about 1nM, about 15pM to about 950pM, about 15pM to about 900pM, about 15pM to about 850pM, about 15pM to about 800pM, about 15pM to about 750pM, about 15pM to about 700pM, about 15pM to about 650pM, about 15pM to about 600pM, about 15pM to about 550pM, about 15pM to about 500pM, about 15pM to about 450pM, about 15pM to about 400pM, about 15pM to about 350pM, about 15pM to about 300pM, about 15pM to about 250pM, about 15pM to about 200pM, about 15pM to about 150pM, about 15pM to about 100pM, about 15pM to about 90pM, about, about 15pM to about 80pM, about 15pM to about 70pM, about 15pM to about 60pM, about 15pM to about 50pM, about 15pM to about 40pM, about 15pM to about 30pM, about 15pM to about 20pM, about 20pM to about 30nM, about 20pM to about 25nM, about 20pM to about 20nM, about 20pM to about 15nM, about 20pM to about 10nM, about 20pM to about 5nM, about 20pM to about 2nM, about 20pM to about 1nM, about 20pM to about 950pM, about 20pM to about 900pM, about 20pM to about 850pM, about 20pM to about 800pM, about 20pM to about 750pM, about 20pM to about 700pM, about 20pM to about 650pM, about 20pM to about 600pM, about 20pM to about 550pM, about 20pM to about 500pM, about 20pM to about 450pM, about 20pM to about 400pM, about 20pM to about 350pM, about 20pM to about 300pM, about 20pM to about 250pM, about 20pM to about 20pM, about 200pM to about 150pM, about 20pM to about 100pM, about 20pM to about 90pM, about 20pM to about 80pM, about 20pM to about 70pM, about 20pM to about 60pM about 20pM to about 50pM, about 20pM to about 40pM, about 20pM to about 30pM, about 30pM to about 30nM, about 30pM to about 25nM, about 30pM to about 30nM, about 30pM to about 15nM, about 30pM to about 10nM, about 30pM to about 5nM, about 30pM to about 2nM, about 30pM to about 1nM, about 30pM to about 950pM, about 30pM to about 900pM, about 30pM to about 850pM, about 30pM to about 800pM, about 30pM to about 750pM, about 30pM to about 700pM, about 30pM to about 650pM, about 30pM to about 600pM, about 30pM to about 550pM, about 30pM to about 500pM about 30pM to about 450pM, about 30pM to about 400pM, about 30pM to about 350pM, about 30pM to about 300pM, about 30pM to about 250pM, about 30pM to about 200pM, about 30pM to about 150pM, about 30pM to about 100pM, about 30pM to about 90pM, about 30pM to about 80pM, about 30pM to about 70pM, about 30pM to about 60pM, about 30pM to about 50pM, about 30pM to about 40pM, about 40pM to about 30nM, about 40pM to about 25nM, about 40pM to about 30nM, about 40pM to about 15nM, about 40pM to about 10nM, about 40pM to about 5nM, about 40pM to about 2nM, about 40pM to about 1nM about 40pM to about 950pM, about 40pM to about 900pM, about 40pM to about 850pM, about 40pM to about 800pM, about 40pM to about 750pM, about 40pM to about 700pM, about 40pM to about 650pM, about 40pM to about 600pM, about 40pM to about 550pM, about 40pM to about 500pM, about 40pM to about 450pM, about 40pM to about 400pM, about 40pM to about 350pM, about 40pM to about 300pM, about 40pM to about 250pM, about 40pM to about 200pM, about 40pM to about 150pM, about 40pM to about 100pM, about 40pM to about 90pM, about 40pM to about 80pM, about 40pM to about 70pM, about, about 40pM to about 60pM, about 40pM to about 50pM, about 50pM to about 30nM, about 50pM to about 25nM, about 50pM to about 30nM, about 50pM to about 15nM, about 50pM to about 10nM, about 50pM to about 5nM, about 50pM to about 2nM, about 50pM to about 1nM, about 50pM to about 950pM, about 50pM to about 900pM, about 50pM to about 850pM, about 50pM to about 800pM, about From 50pM to about 750pM, from about 50pM to about 700pM, from about 50pM to about 650pM, from about 50pM to about 600pM, from about 50pM to about 550pM, from about 50pM to about 500pM, from about 50pM to about 450pM, from about 50pM to about 400pM, from about 50pM to about 350pM, from about 50pM to about 300pM, from about 50pM to about 250pM, from about 50pM to about 200pM, from about 50pM to about 150pM, from about 50pM to about 100pM, from about 50pM to about 90pM, from about 50pM to about 80pM, from about 50pM to about 70pM, from about 50pM to about 60pM, from about 60pM to about 30nM, from about 60pM to about 25nM, from about 60pM to about 30nM, from about 60pM to about 15nM about 60pM to about 10nM, about 60pM to about 5nM, about 60pM to about 2nM, about 60pM to about 1nM, about 60pM to about 950pM, about 60pM to about 900pM, about 60pM to about 850pM, about 60pM to about 800pM, about 60pM to about 750pM, about 60pM to about 700pM, about 60pM to about 650pM, about 60pM to about 600pM, about 60pM to about 550pM, about 60pM to about 500pM, about 60pM to about 450pM, about 60pM to about 400pM, about 60pM to about 350pM, about 60pM to about 300pM, about 60pM to about 250pM, about 60pM to about 200pM, about 60pM to about 150pM about 60pM to about 100pM, about 60pM to about 90pM, about 60pM to about 80pM, about 60pM to about 70pM, about 70pM to about 30nM, about 70pM to about 25nM, about 70pM to about 30nM, about 70pM to about 15nM, about 70pM to about 10nM, about 70pM to about 5nM, about 70pM to about 2nM, about 70pM to about 1nM, about 70pM to about 950pM, about 70pM to about 900pM, about 70pM to about 850pM, about 70pM to about 800pM, about 70pM to about 750pM, about 70pM to about 700pM, about 70pM to about 650pM, about 70pM to about 600pM, about 70pM to about 550pM, about 70pM to about 500pM about 70pM to about 450pM, about 70pM to about 400pM, about 70pM to about 350pM, about 70pM to about 300pM, about 70pM to about 250pM, about 70pM to about 200pM, about 70pM to about 150pM, about 70pM to about 100pM, about 70pM to about 90pM, about 70pM to about 80pM, about 80pM to about 30nM, about 80pM to about 25nM, about 80pM to about 30nM, about 80pM to about 15nM, about 80pM to about 10nM, about 80pM to about 5nM, about 80pM to about 2nM, about 80pM to about 1nM, about 80pM to about 950pM, about 80pM to about 900pM, about 80pM to about 850pM, about 80pM to about, about 80pM to about 800pM, about 80pM to about 750pM, about 80pM to about 700pM, about 80pM to about 650pM, about 80pM to about 600pM, about 80pM to about 550pM, about 80pM to about 500pM, about 80pM to about 450pM, about 80pM to about 400pM, about 80pM to about 350pM, about 80pM to about 300pM, about 80pM to about 250pM, about 80pM to about 200pM, about 80pM to about 1 pM 50pM, about 80pM to about 100pM, about 80pM to about 90pM, about 90pM to about 30nM, about 90pM to about 25nM, about 90pM to about 30nM, about 90pM to about 15nM, about 90pM to about 10nM, about 90pM to about 5nM, about 90pM to about 2nM, about 90pM to about 1nM, about 90pM to about 950pM, about 90pM to about 900pM, about 90pM to about 850pM, about 90pM to about 800pM, about 90pM to about 750pM, about 90pM to about 700pM, about 90pM to about 650pM, about 90pM to about 600pM, about 90pM to about 550pM, about 90pM to about 500pM, about 90pM to about 450pM, about 90pM to about 400pM about 90pM to about 350pM, about 90pM to about 300pM, about 90pM to about 250pM, about 90pM to about 200pM, about 90pM to about 150pM, about 90pM to about 100pM, about 100pM to about 30nM, about 100pM to about 25nM, about 100pM to about 30nM, about 100pM to about 15nM, about 100pM to about 10nM, about 100pM to about 5nM, about 100pM to about 2nM, about 100pM to about 1nM, about 100pM to about 950pM, about 100pM to about 900pM, about 100pM to about 850pM, about 100pM to about 800pM, about 100pM to about 750pM, about 100pM to about 700pM, about 100pM to about 650pM, about 100pM to about 750pM about 100pM to about 600pM, about 100pM to about 550pM, about 100pM to about 500pM, about 100M to about 450pM, about 100pM to about 400pM, about 100pM to about 350pM, about 100pM to about 300pM, about 100pM to about 250pM, about 100pM to about 200pM, about 100pM to about 150pM, about 150pM to about 30nM, about 150pM to about 25nM, about 150pM to about 30nM, about 150pM to about 15nM, about 150pM to about 10nM, about 150pM to about 5nM, about 150pM to about 2nM, about 150pM to about 1nM, about 150pM to about 950pM, about 150pM to about 900pM, about 150pM to about 850pM, about 150pM to about 800pM about 150pM to about 750pM, about 150pM to about 700pM, about 150pM to about 650pM, about 150pM to about 600pM, about 150pM to about 550pM, about 150pM to about 500pM, about 150pM to about 450pM, about 150pM to about 400pM, about 150pM to about 350pM, about 150pM to about 300pM, about 150pM to about 250pM, about 150pM to about 200pM, about 200pM to about 30nM, about 200pM to about 25nM, about 200pM to about 30nM, about 200pM to about 15nM, about 200pM to about 10nM, about 200pM to about 5nM, about 200pM to about 2nM, about 200pM to about 1nM, about 200pM to about 950pM, about 200pM to about 900pM, about 200pM to about 850pM, about 200pM to about 800pM, about 200pM to about 750pM, about 200pM to about 700pM, about 200pM to about 650pM, about 200pM to about 600pM, about 200pM to about 550pM, about 200pM to about 500pM, about 200pM to about 450pM, about 200pM to about 400pM, about 200pM to about 350pM, about 200pM to about 300pM, about 200pM to about 250pM, about 300pM to about 30nM, about 300pM to about 25nM, about 300pM to about 30nM, about 300pM to about 15nM, about 300pM to about 10nM, about 300pM to about 5nM, about 300pM to about 2nM, about 300pM to about 1nM, about 300pM to about 950pM, about 300pM to about 900pM, about 300pM to about 850pM, about 300pM to about 800pM, about 300pM to about 750pM, about 300pM to about 700pM, about 300pM to about 650pM, about 300pM to about 600pM, about 300pM to about 550pM, about 300pM to about 500pM about 300pM to about 450pM, about 300pM to about 400pM, about 300pM to about 350pM, about 400pM to about 30nM, about 400pM to about 25nM, about 400pM to about 30nM, about 400pM to about 15nM, about 400pM to about 10nM, about 400pM to about 5nM, about 400pM to about 2nM, about 400pM to about 1nM, about 400pM to about 950pM, about 400pM to about 900pM, about 400pM to about 850pM, about 400pM to about 800pM, about 400pM to about 750pM, about 400pM to about 700pM, about 400pM to about 650pM, about 400pM to about 600pM, about 400pM to about 550pM, about 400pM to about 500pM about 500pM to about 30nM, about 500pM to about 25nM, about 500pM to about 30nM, about 500pM to about 15nM, about 500pM to about 10nM, about 500pM to about 5nM, about 500pM to about 2nM, about 500pM to about 1nM, about 500pM to about 950pM, about 500pM to about 900pM, about 500pM to about 850pM, about 500pM to about 800pM, about 500pM to about 750pM, about 500pM to about 700pM, about 500pM to about 650pM, about 500pM to about 600pM, about 500pM to about 550pM, about 600pM to about 30nM, about 600pM to about 25nM, about 600pM to about 30nM, about 600pM to about 15nM, about 600pM to about 10nM about 600pM to about 5nM, about 600pM to about 2nM, about 600pM to about 1nM, about 600pM to about 950pM, about 600pM to about 900pM, about 600pM to about 850pM, about 600pM to about 800pM, about 600pM to about 750pM, about 600pM to about 700pM, about 600pM to about 650pM, about 700pM to about 30nM, about 700pM to about 25nM, about 700pM to about 30nM, about 700pM to about 15nM, about 700pM to about 10nM, about 700pM to about 5nM, about 700pM to about 2nM, about 700pM to about 1nM, about 700pM to about 950pM, about 700pM to about 900pM, about 700pM to about 850pM, about, about 700pM to about 800pM, about 700pM to about 750pM, about 800pM to about 30nM, about 800pM to about 25nM, about 800pM to about 30nM, about 800pM to about 15nM, about 800pM to about 10nM, about 800pM to about 5nM, about 800pM to about 2nM, about 800pM to about 1nM, about 80 nM 0pM to about 950pM, about 800pM to about 900pM, about 800pM to about 850pM, about 900pM to about 30nM, about 900pM to about 25nM, about 900pM to about 30nM, about 900pM to about 15nM, about 900pM to about 10nM, about 900pM to about 5nM, about 900pM to about 2nM, about 900pM to about 1nM, about 900pM to about 950pM, about 1nM to about 30nM, about 1nM to about 25nM, about 1nM to about 20nM, about 1nM to about 15nM, about 1nM to about 10nM, about 1nM to about 5nM, about 2nM to about 30nM, about 2nM to about 25nM, about 2nM to about 20nM, about 2nM to about 15nM about 2nM to about 10nM, about 2nM to about 5nM, about 4nM to about 30nM, about 4nM to about 25nM, about 4nM to about 20nM, about 4nM to about 15nM, about 4nM to about 10nM, about 4nM to about 5nM, about 5nM to about 30nM, about 5nM to about 25nM, about 5nM to about 20nM, about 5nM to about 15nM, about 5nM to about 10nM, about 10nM to about 30nM, about 10nM to about 25nM, about 10nM to about 20nM, about 10nM to about 15nM, about 15nM to about 30nM, about 15nM to about 25nM, about 15nM to about 20nM, about 20nM to about 30nM, and about 20nM to about 25 nM) _D To bind to its target.

Any of the target binding domains described herein can be between about 1nM and about 10nM (e.g., about 1nM to about 9nM, about 1nM to about 8nM, about 1nM to about 7nM, about 1nM to about 6nM, about 1nM to about 5nM, about 1nM to about 4nM, about 1nM to about 3nM, about 1nM to about 2nM, about 2nM to about 10nM, about 2nM to about 9nM, about 2nM to about 8nM, about 2nM to about 7nM, about 2nM to about 6nM, about 2nM to about 5nM, about 2nM to about 4nM, about 2nM to about 3nM, about 3nM to about 10nM, about 3nM to about 9nM, about 3nM to about 8nM, about 3nM to about 7nM, about 3nM to about 6nM, about 3nM to about 5nM about 3nM to about 4nM, about 4nM to about 10nM, about 4nM to about 9nM, about 4nM to about 8nM, about 4nM to about 7nM, about 4nM to about 6nM, about 4nM to about 5nM, about 5nM to about 10nM, about 5nM to about 9nM, about 5nM to about 8nM, about 5nM to about 7nM, about 5nM to about 6nM, about 6nM to about 10nM, about 6nM to about 9nM, about 6nM to about 8nM, about 6nM to about 7nM, about 7nM to about 10nM, about 7nM to about 9nM, about 7nM to about 8nM, about 8nM to about 10nM, about 8nM to about 9nM and about 9nM to about 10 nM) _D To bind to its target.

The K of any of the antigen binding protein constructs described herein can be determined using a variety of different methods known in the art _D Values (e.g. electrophoretic mobility change assayFilter binding assays, surface plasmon resonance and biomolecular binding kinetics assays, etc.).

Antigen binding domains

In some embodiments of any of the single-or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of these single-or multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these single-or multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each an antigen binding domain. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is an scFv or single domain antibody (e.g., va _H H or V _NAR Domain).

In some embodiments, an antigen binding domain (e.g., any of the antigen binding domains described herein) can specifically bind to any of the following: CD16a (see, e.g., those described in U.S. patent No. 9,035,026), CD28 (see, e.g., those described in U.S. patent No. 7,723,482), CD3 (see, e.g., those described in U.S. patent No. 9,226,962), CD33 (see, e.g., those described in U.S. patent No. 8,759,494), CD20 (see, e.g., those described in WO 2014/026054), CD19 (see, e.g., those described in U.S. patent No. 9,701,758), CD22 (see, e.g., those described in WO 2003/104425), CD123 (see, e.g., those described in WO 2014/130635), IL-1R (see, e.g., those described in U.S. patent No. 8,741,604), IL-1 (see, e.g., those described in WO 2014/095808), VEGF (see, e.g., those described in U.S. patent No. 9,090,684), IL-6R (see, e.g., those described in U.S. patent No. 7,482,436), IL-4 (see, e.g., those described in U.S. patent No. 2012, e.S. laid-open patent No. light-7, e.g., those described in patent No. light-load-time window patent No. 37, e.g., patent No. 13, e.g., see, e.g., patent No. 37 f, e.g., patent No. 37, e.g., patent No. 13, e.g., see, e.g., patent publication No. 1, e.g., see, e.g., patent No. 13, e.g., see, e.g., patent publication No. 2011, e.g., see, etc.), p.g., see, e.g., publication No. patent publication No. 2011, see, etc.). Those described in U.S. patent No. 8,552,156), CTLA4 (see, e.g., those described in WO 2012/120125), MICA (see, e.g., those described in WO 2016/154585), MICB (see, e.g., those described in U.S. patent No. 8,753,640), IL-6 (see, e.g., gejima et al, human Antibodies 11 (4): 121-129, 2002), IL-8 (see, e.g., those described in U.S. patent No. 6,117,980), tnfα (see, e.g., geng et al, immunol. Res.62 (3): 377-385, 2015), CD26 (see, e.g., those described in WO 2017/189526), CD36 (see, e.g., those described in U.S. patent application publication No. 2015/0259429), ULBP2 (see, e.g., those described in U.S. patent No. 9,273,136), CD30 (see, e.g., homach et al, scand.j.immunol.48 (5): 497-501, 1998), CD200 (see, e.g., those described in U.S. patent No. 9,085,623), IGF-1R (see, e.g., those described in U.S. patent application publication No. 2017/0051063), MUC4AC (see, e.g., those described in WO 2012/170470), MUC5AC (see, e.g., those described in U.S. patent No. 9,238,084), trop-2 (see, e.g., those described in WO 2013/068946), CMET (see, e.g., edwards raja et al, biotechnol bioeng 106 (3): 367-375, 2010), EGFR (see, e.g., akbari et al, protein expr. Purif.127:8-15, 2016), HER1 (see, e.g., those described in U.S. patent application publication No. 2013/0274446), HER2 (see, e.g., cao et al, biotechnol lett.37 (7): 1347-1354, 2015), HER3 (see, e.g., those described in U.S. patent No. 9,505,843), PSMA (see, e.g., parker et al Protein expr. Purif.89 (2): 136-145, 2013), CEA (see, e.g., those described in WO 1995/015341), B7H3 (see, e.g., those described in U.S. patent No. 9,371,395), EPCAM (see, e.g., those described in WO 2014/159531), BCMA (see, e.g., smith et al, mol. Ter. 26 (6): 1447-1456, 2018), P-cadherin (see, e.g., those described in U.S. patent No. 7,452,537), CEACAM5 (see, e.g., those described in U.S. patent No. 9,617,345), UL16 binding protein (see, e.g., those described in WO 2017/083612), HLA-DR (see, e.g., pistillo et al exp.clin.immunogene.14 (2), and (ii) the like: 123-130, 1997), DLL4 (see, e.g., those described in WO 2014/007513), TYRO3 (see, e.g., those described in WO 2016/166348), AXL (see, e.g., those described in WO 2012/175692), MER (see, e.g., those described in WO 2016/106221), CD122 (see, e.g., those described in U.S. patent application publication No. 2016/036764), CD155 (see, e.g., those described in WO 2017/149538), or PDGF-DD (see, e.g., those described in U.S. patent No. 9,441,034).

The antigen binding domains present in any of the single-chain chimeric polypeptides or multi-chain chimeric polypeptides described herein are each independently selected from the group consisting of: VHH domain, VNAR domain, and scFv. In some embodiments, any of the antigen binding domains described herein are BiTe, (scFv) ₂ Nanobody, nanobody-HAS, DART, tandAb, scDiabody-CH 3, scFv-CH-CL-scFv, hsambody, single chain bifunctional antibody-HAS, or tandem scFv. Additional examples of antigen binding domains that can be used in any single or multi-chain chimeric polypeptide are known in the art.

VHH domains are single monomer variable antibody domains that can be found in camelids. The VNAR domain is a single monomeric antibody variable domain that can be found in cartilaginous fish. V (V) _H H domain and V _NAR Non-limiting aspects of the domains are described, for example, in cronie et al, curr. Top. Med. Chem.15:2543-2557, 2016; de Genst et al, dev. Comp. Immunol.30:187-198, 2006; de Meyer et al, trends Biotechnol.32:263-270, 2014; kijanka et al, nanomedicine 10:161-174, 2015; kovaleva et al, experert.opin.biol.Ther.14: 1527-1539, 2014; krah et al, immunology. 38:21-28, 2016; mujic-Delic et al, trends Pharmacol. Sci.35:247-255, 2014; muydermans, j.biotechnol.74:277-302, 2001; muydermans et al, trends biochem. Sci.26:230-235, 2001; muydermans, ann.rev.biochem.82:775-797, 2013; rahbarizadeh et al, immunol. Invest.40:299-338, 2011; van Audenhove et al, EBiomedicine 8:40-48, 2016; van Bockstaele et al, curr. Opin. Invest. Drugs 10:1212-1224, 2009; vincke et al, methods mol. Biol.911:15-26, 2012; and Wesolowski et al, med. Microbiol. Immunol.198:157-174, 2009.

In some embodiments, each antigen binding domain in a single-chain or multi-chain chimeric polypeptide described herein is a VHH domain, or at least one antigen binding domain is a VHH domain. In some embodiments, each antigen binding domain in a single-chain or multi-chain chimeric polypeptide described herein is a VNAR domain, or at least one antigen binding domain is a VNAR domain. In some embodiments, each antigen binding domain in a single-chain or multi-chain chimeric polypeptide described herein is an scFv domain, or at least one antigen binding domain is an scFv domain.

In some embodiments, two or more polypeptides present in a single-chain or multi-chain chimeric polypeptide mayAny of the antigen binding domains described herein, e.g., antigen binding fragments of antibodies (e.g., any of the antigen binding fragments of antibodies described herein), VHH-scabs, VHH-fabs, bisfab, F (ab ') 2, bifunctional antibodies, interchangeable monoclonal antibodies, DAF (two-in-one), DAF (four-in-one), dutaMab, DT-IgG, mortar-pestle type common light chain, mortar-pestle type assemblies, charge pairs, fab arm exchanges, SEEDbody, LUZ-Y, fcab, kappa lambda, orthogonal Fab, DVD-IgG, igG (H) -scFv, scFv- (H) IgG, igG (L) -scFv, scFv- (L) IgG, igG (L), H) -Fv, igG (H) -V, V (H) -IgG, igG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, igG-2scFv, scFv4-Ig, zybody, DVI-IgG, diabody-CH 3, triad body, minibody, triBi minibody, scFv-CH3 KIH, fab-scFv, F (ab') 2-scFv2, scFv-KIH, fab-scFv-Fc, tetravalent HCAb, single chain diabody-Fc, tandem scFv-Fc, intracellular antibody, docking and locking (dock and lock), lmTAC, igG-IgG conjugates, cov-X body, and scFv1-PEG-scFv2. See, e.g., spiess et al, mol. Immunol.67:95-106, 2015, which are incorporated herein in their entirety to describe these elements. Non-limiting examples of antigen binding fragments of antibodies include Fv fragments, fab fragments, F (ab') ₂ Fragments and Fab' fragments. Further examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, igG2, igG3, or IgG 4) (e.g., human or humanized IgG, such as antigen-binding fragments of human or humanized IgG1, igG2, igG3, or IgG 4); an antigen-binding fragment of IgA (e.g., an antigen-binding fragment of IgA1 or IgA 2) (e.g., a human or humanized IgA, such as an antigen-binding fragment of human or humanized IgA1 or IgA 2); antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD); antigen binding fragments of IgE (e.g., antigen binding fragments of human or humanized IgE); or an antigen-binding fragment of IgM (e.g., an antigen-binding fragment of human or humanized IgM).

An "Fv" fragment comprises a non-covalently linked dimer of one heavy chain variable domain and one light chain variable domain.

"Fab" fragments includeIn addition to the heavy and light chain variable domains of Fv fragments, the constant domain of the light chain and the first constant domain of the heavy chain (C _H1 )。

“F(ab') ₂ "fragments include two Fab fragments joined by a disulfide bond near the hinge region.

"Dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and multispecific binding proteins, such as, for example, digiammarino et al, methods mol. Biol.899:145-156, 2012; jakob et al, MABs 5:358-363, 2013; and U.S. patent No. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, each of which is incorporated by reference in its entirety.

DART is described, for example, in Garber, nature Reviews Drug Discovery 13:799-801,2014.

In some embodiments, any of the antigen binding domains described herein are capable of binding to an antigen selected from the group consisting of: proteins, carbohydrates, lipids, and combinations thereof.

Other embodiments and aspects of antigen binding domains are known in the art.

Soluble interleukin or cytokine proteins

In some embodiments of any of the single-or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain can be a soluble interleukin protein or a soluble cytokine protein. In some embodiments, the soluble interleukin or soluble cytokine protein is selected from the group consisting of: IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF are provided below.

Human soluble IL-3 (SEQ ID NO: 105)

Human soluble IL-8 (SEQ ID NO: 106)

Human soluble IL-10 (SEQ ID NO: 107)

Human soluble IL-17 (SEQ ID NO: 108)

Human soluble IL-18 (SEQ ID NO: 109)

Human soluble PDGF-DD (SEQ ID NO: 110)

Human soluble SCF (SEQ ID NO: 111)

Human soluble FLT3L (SEQ ID NO: 112)

Additional examples of soluble interleukin proteins and soluble cytokine proteins are known in the art.

Soluble receptors

In some embodiments of any of the single-or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor or a soluble cytokine receptor. In some embodiments, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII) (see, e.g., yung et al, am. J. Resp. Crit. Care Med.194 (9): 1140-1151, 2016), soluble TGF-beta RIII (see, e.g., heng et al, plamenta 57:320, 2017), soluble NKG2D (see, e.g., cosman et al, immunity 14 (2): 123-133, 2001; costa et al, front. Immunol., volume 9, strip 1150, 2018, day 5, 29; doi: 10.3389/fimmu.2018.01150), soluble NKp30 (see, e.g., costa et al, front. Immunol., volume 9, 1150, 2018, 5, 29; doi: 10.3389/fimmu.2018.01150), soluble NKp44 (see, e.g., costa et al, front. Immunol., volume 9, 1150, 2018, 5, 29; doi: 10.3389/fimmu.2018.01150), soluble NKp46 (see, e.g., those described in Mandelboim et al, nature 409:1055-1060, 2001; costa et al, front. Immunol., volume 9, strip 1150, 2018, month 29; doi: 10.3389/fimmu.2018.01150), soluble DNAM1 (see, e.g., costa et al, front. Immunol., volume 9, strip 1150, 2018, month 29; doi: 10.3389/fimmu.2018.01150), scMHI (see, e.g., those described in Washburn et al, PLoS On 6 (3): e18439, 2011), scMHI (see, e.g., bishwajit et al, cell. 170 (1): 25-33, 1996), and (see, e.g., those described in Weber, 1996, 199356, etc.) Soluble CD155 (see, e.g., tahara-Hanaoka et al, int. Immunol.16 (4): 533-538, 2004) or soluble CD28 (see, e.g., hebbar et al, clin. Exp. Immunol.136:388-392, 2004).

Other examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

Affinity domain pairs

In some embodiments, the multi-chain chimeric polypeptide comprises: 1) A first chimeric polypeptide comprising a first domain in a pair of affinity domains, and 2) a second chimeric polypeptide comprising a second domain in a pair of affinity domains, such that the first chimeric polypeptide and the second chimeric polypeptide associate by binding of the first domain and the second domain in the pair of affinity domains. In some embodiments, the pair of affinity domains is a sushi domain from the alpha chain of the human IL-15 receptor (IL 15Rα) and soluble IL-15.sushi domains, also known as short consensus repeats or glycoprotein type 1 motifs, are common motifs in protein-protein interactions. Sushi domains have been identified on a number of protein binding molecules, including complement components C1r, C1s, factor H and C2m, and the nonimmunosolecules factor XIII and β2-glycoprotein. A typical sushi domain has about 60 amino acid residues and comprises four cysteines (Ranganathan, pac. Symp Biocomputer.2000:155-67). The first cysteine may form a disulfide bond with the third cysteine, and the second cysteine may form a disulfide bridge with the fourth cysteine. In some embodiments in which one member of a pair of affinity domains is soluble IL-15, soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments in which one member of a pair of affinity domains is the alpha chain of the human IL-15 receptor (IL 15 ra), the human IL15 ra is mature full length IL15 ra. In some embodiments, a pair of affinity domains is bacillus ribonuclease and bacillus ribonuclease inhibitor. In some embodiments, the pair of affinity domains are PKA and AKAP. In some embodiments, the pair of affinity domains are adaptor/docking tag modules based on mutant ribonuclease I fragments (Rossi, proc Natl Acad Sci USA.103:6841-6846, 2006; sharkey et al, cancer Res.68:5282-5290, 2008; rossi et al, trends Pharmacol Sci.33:474-481, 2012) or SNARE modules based on interaction of protein synaptotagins, synaptotagins and SNAP25 (Deyev et al, nat Biotechnol.1486-1492, 2003).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide comprises a first domain in a pair of affinity domains, and a second chimeric polypeptide of a multi-chain chimeric polypeptide comprises a second domain in a pair of affinity domains, wherein the first domain in the pair of affinity domainsThe domain and the second domain of the pair of affinity domains are less than 1x10 ^-7 M is less than 1x10 ^-8 M is less than 1x10 ^-9 M is less than 1x10 ^-10 M is less than 1x10 ^-11 M is less than 1x10 ^-12 M or less than 1x10 ^-13 Dissociation equilibrium constant of M (K _D ) Are combined with each other. In some embodiments, the first domain in the pair of affinity domains and the second domain in the pair of affinity domains are at about 1x10 ^-4 M to about 1x10 ^-6 M, about 1x10 ^-5 M to about 1x10 ^-7 M, about 1x10 ^{- 6} M to about 1x10 ^-8 M, about 1x10 ^-7 M to about 1x10 ^-9 M, about 1x10 ^-8 M to about 1x10 ^-10 M, about 1x10 ^-9 M to about 1x10 ^-11 M, about 1x10 ^-10 M to about 1x10 ^-12 M, about 1x10 ^-11 M to about 1x10 ^-13 M, about 1x10 ^-4 M to about 1x10 ^-5 M, about 1x10 ^-5 M to about 1x10 ^{- 6} M, about 1x10 ^-6 M to about 1x10 ^-7 M, about 1x10 ^-7 M to about 1x10 ^-8 M, about 1x10 ^-8 M to about 1x10 ^-9 M, about 1x10 ^-9 M to about 1x10 ^-10 M, about 1x10 ^-10 M to about 1x10 ^-11 M, about 1x10 ^-11 M to about 1x10 ^-12 M or about 1x10 ^-12 M to about 1x10 ^-13 K of M _D Are combined with each other. Any of a number of different methods known in the art may be used to determine the K binding of a first domain in a pair of affinity domains to a second domain in a pair of affinity domains _D Values (e.g., electrophoretic migration variation assay, filter binding assay, surface plasmon resonance, and biomolecule binding kinetics assay, etc.).

In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide comprises a first domain of a pair of affinity domains and a second chimeric polypeptide of a multi-chain chimeric polypeptide comprises a second domain of a pair of affinity domains, wherein the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both are about 10 to 100 amino acids in length. For example, the number of the cells to be processed, the first domain in the pair of affinity domains, the second domain in the pair of affinity domains, or both, may be about 10 to 100 amino acids in length, may be about 15 to 100 amino acids in length, may be about 20 to 100 amino acids in length, may be about 25 to 100 amino acids in length, may be about 30 to 100 amino acids in length, may be about 35 to 100 amino acids in length, may be about 40 to 100 amino acids in length, may be about 45 to 100 amino acids in length, may be about 50 to 100 amino acids in length, may be about 55 to 100 amino acids in length, may be about 60 to 100 amino acids in length, may be about 65 to 100 amino acids in length, may be about 70 to 100 amino acids in length, may be about 75 to 100 amino acids in length, may be about 80 to 100 amino acids in length, may be about 85 to 100 amino acids in length about 90 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 amino acids in length, about 10 to 75 amino acids in length, about 10 to 70 amino acids in length, about 10 to 65 amino acids in length, about 10 to 60 amino acids in length, about 10 to 55 amino acids in length, about 10 to 50 amino acids in length, about 10 to 45 amino acids in length, about 10 to 40 amino acids in length, about 10 to 35 amino acids in length, about 10 to 20 amino acids in length, about 10 to 15 amino acids in length, the length may be about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the first domain in a pair of affinity domains, the second domain in a pair of affinity domains, or both are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the first and/or second domains of a pair of affinity domains disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, so long as the function of the first and/or second domain of the pair of affinity domains remains intact. For example, the sushi domain of the alpha chain (il15rα) from the human IL-15 receptor may include one or more additional amino acids at the N-terminus and/or C-terminus, while still maintaining the ability to bind to soluble IL-15. Additionally or alternatively, the soluble IL-15 may comprise one or more additional amino acids at the N-terminus and/or the C-terminus, while still retaining the ability to bind to the sushi domain of the human IL-15 receptor alpha chain (IL-15 ra).

Non-limiting examples of sushi domains from the alpha chain of IL-15 receptor alpha (IL 15 Ralpha) may include sequences that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 113). In some embodiments, the sushi domain from the alpha chain of IL-15Rα may be defined by a sequence comprising

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 114).

In some embodiments, soluble IL-15 may include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 115). In some embodiments, soluble IL-15 may be encoded by a nucleic acid comprising the sequence of AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 116).

Signal sequence

In some embodiments, the single-chain chimeric polypeptide comprises a signal sequence at its N-terminus. In some embodiments, the multi-chain chimeric polypeptide comprises a first chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, the multi-chain chimeric polypeptide comprises a second chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide both comprise a signal sequence. As will be appreciated by those skilled in the art, a signal sequence is an amino acid sequence that is present at the N-terminus of many endogenously produced proteins that directs the protein to the secretory pathway (e.g., the protein is directed to be present in certain intracellular organelles, present in the cell membrane, or secreted from the cell). The signal sequences are heterogeneous and their primary amino acid sequences vary widely. However, the signal sequence is typically 16 to 30 amino acids in length and includes a hydrophilic, usually positively charged, N-terminal region, a central hydrophobic binding domain, and a C-terminal region containing a cleavage site for a signal peptidase.

In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single chain chimeric polypeptide, comprises a signal sequence having amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 117). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide, comprises a polypeptide sequence consisting of nucleic acid sequence ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 118),

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 119) or

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 120).

In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single chain chimeric polypeptide, comprises a signal sequence having amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 121). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single chain chimeric polypeptide, comprises a signal sequence having amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 122). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single chain chimeric polypeptide, comprises a signal sequence having amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 123). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single chain chimeric polypeptide, comprises a signal sequence having amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 124). Those of skill in the art will appreciate other suitable signal sequences for the first chimeric polypeptide and/or the second chimeric polypeptide, or the single chain chimeric polypeptides, used in the multi-chain chimeric polypeptides described herein.

In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide comprises a signal sequence of about 10 to 100 amino acids in length. For example, the number of the cells to be processed, the signal sequence may be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length about 10 to about 90 amino acids in length, about 10 to about 85 amino acids in length, about 10 to about 80 amino acids in length, about 10 to about 75 amino acids in length, about 10 to about 70 amino acids in length, about 10 to about 65 amino acids in length, about 10 to about 60 amino acids in length, about 10 to about 55 amino acids in length, about 10 to about 50 amino acids in length, about 10 to about 45 amino acids in length, about 10 to about 40 amino acids in length, about 10 to about 35 amino acids in length, about 10 to about 30 amino acids in length, about 10 to about 25 amino acids in length, about 10 to about 20 amino acids in length, about 20 to about 30 amino acids in length, about 30 to about 40 amino acids in length, the length may be about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the signal sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

In some embodiments, any of the signal sequences disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and/or C-terminus, so long as the function of the signal sequence remains intact. For example, a signal sequence having amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 125) may include one or more additional amino acids at the N-terminus or the C-terminus while still retaining the ability to direct the first chimeric polypeptide in a multi-chain chimeric polypeptide, the second chimeric polypeptide in a multi-chain chimeric polypeptide, or both, or a single-chain chimeric polypeptide to the secretory pathway.

In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide, a second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide comprises a signal sequence that directs the multi-chain chimeric polypeptide into the extracellular space. Such embodiments are useful for producing single-chain or multi-chain chimeric polypeptides that are relatively easy to isolate and/or purify.

Peptide tag

In some embodiments, the single-chain chimeric polypeptide comprises a peptide tag (e.g., located at the N-terminus or C-terminus of the chimeric polypeptide). In some embodiments, the multi-chain chimeric polypeptide comprises a first chimeric polypeptide comprising a peptide tag (e.g., located at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, the multi-chain chimeric polypeptide comprises a second chimeric polypeptide comprising a peptide tag (e.g., at the N-terminus or C-terminus of the second chimeric polypeptide). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide each comprise a peptide tag. In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide comprises two or more peptide tags.

Exemplary peptide tags that can be incorporated into the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide include, but are not limited to, aviTag (GLNDIFEAQKIEWHE; SEQ ID NO: 126), calmodulin tag (GLNDIFEAQKIEWHE; SEQ ID NO: 127), polyglutamate tag (EEEEEE; SEQ ID NO: 128), E tag (GAPVPYPDPLEPR; SEQ ID NO: 129), FLAG tag (DYKDDDK; SEQ ID NO: 130), HA tag, peptide from hemagglutinin (YPVPDDYA; SEQ ID NO: 131), his tag (HHHHH (SEQ ID NO: 132), HHHHH (SEQ ID NO: 133), HHHHHHHH (SEQ ID NO: 134), HHHH (SEQ ID NO: 135), HHH HHHHHHHHH (SEQ ID NO: 136), or HHHHHHHHHH (SEQ ID NO: 137)), myc tag (EQKLISEEDL, NE (52; 52), FLAG tag (DKDDDDDDDDDK; SEQ ID NO: 130), HA tag, peptide (YPdVPDDdDYA; 131), his tag (HHHHH (SEQ ID NO: 134), HHHH (SEQ ID NO: 135), HHHHH tag (SEQ ID NO: 133), HHHHH tag (SEQ ID NO: 95), HHHHH (SEQ ID NO: 35), or HHHHH tag (SEQ ID NO: 136), or WSID tag (SEQ ID NO: 35), and Xpress tag (DLYDDDDK; SEQ ID NO: 150). In some embodiments, the tissue factor protein is a peptide tag.

The peptide tag of the first chimeric polypeptide capable of incorporating the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide, respectively, can be used in any of a variety of applications related to multi-chain or single-chain chimeric polypeptides. For example, the peptide tags can be used to purify a multi-chain or single-chain chimeric polypeptide. As one non-limiting example, a first chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a first chimeric polypeptide expressed recombinantly), a second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a second chimeric polypeptide expressed recombinantly), or both, or the single-chain chimeric polypeptide may include a myc tag; a multi-chain chimeric polypeptide comprising a first chimeric polypeptide labeled with myc, a second chimeric polypeptide labeled with myc, or both, or a single-chain chimeric polypeptide labeled with myc can be purified using an antibody that recognizes a myc tag. One non-limiting example of an antibody that recognizes the myc tag is 9E10, which is available from non-commercial development research hybridoma libraries (Developmental Studies Hybridoma Bank). As another non-limiting example, a first chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a first chimeric polypeptide expressed recombinantly), a second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a second chimeric polypeptide expressed recombinantly), or both, or the single-chain chimeric polypeptide may include a histidine tag; a multi-chain chimeric polypeptide comprising a first chimeric polypeptide tagged with histidine, a second chimeric polypeptide tagged with histidine, or both, or a single-chain chimeric polypeptide tagged with histidine can be purified using a nickel or cobalt chelator. Those of ordinary skill in the art will appreciate other suitable tags and agents that bind those tags for purification of single-or multi-chain chimeric polypeptides. In some embodiments, after purification, the peptide tag is removed from the first chimeric polypeptide and/or the second chimeric polypeptide in the multi-chain chimeric polypeptide or from the single-chain chimeric polypeptide. In some embodiments, the peptide tag is not removed from the first chimeric polypeptide and/or the second chimeric polypeptide or the single chain chimeric polypeptide in the multi-chain chimeric polypeptide after purification.

The peptide tag of the first chimeric polypeptide, the second chimeric polypeptide, or both, or the single chain chimeric polypeptide, which is capable of incorporating the multi-chain chimeric polypeptide, can be used, for example, for immunoprecipitation of the multi-chain chimeric polypeptide or single chain chimeric polypeptide, respectively, for imaging of the multi-chain chimeric polypeptide or single chain chimeric polypeptide, respectively (e.g., via western blotting, ELISA, flow cytometry, and/or immunocytochemistry), and/or for solubilization of the multi-chain chimeric polypeptide or single chain chimeric polypeptide, respectively.

In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide comprises a peptide tag of about 10 to 100 amino acids in length. For example, the number of the cells to be processed, the peptide tag can be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length about 10 to about 90 amino acids in length, about 10 to about 85 amino acids in length, about 10 to about 80 amino acids in length, about 10 to about 75 amino acids in length, about 10 to about 70 amino acids in length, about 10 to about 65 amino acids in length, about 10 to about 60 amino acids in length, about 10 to about 55 amino acids in length, about 10 to about 50 amino acids in length, about 10 to about 45 amino acids in length, about 10 to about 40 amino acids in length, about 10 to about 35 amino acids in length, about 10 to about 30 amino acids in length, about 10 to about 25 amino acids in length, about 10 to about 20 amino acids in length, about 20 to about 30 amino acids in length, about 30 to about 40 amino acids in length, the length may be about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the peptide tag is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

The peptide tag incorporated into the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide may have any suitable length. For example, the peptide tag can have a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In embodiments where the single or multi-chain chimeric polypeptide comprises two or more peptide tags, the two or more peptide tags may have the same or different lengths. In some embodiments, any of the peptide tags disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminus and/or the C-terminus, so long as the function of the peptide tag remains intact. For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 138) may include one or more additional amino acids (e.g., at the N-terminus and/or C-terminus of the peptide tag) while still retaining the ability to be bound by an antibody (e.g., 9E 10).

Exemplary embodiments of Single chain chimeric polypeptides of type A

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and/or the second target binding domain can independently specifically bind to CD3 (e.g., human CD 3) or CD28 (e.g., human CD 28). In some embodiments, the first target binding domain specifically binds to CD3 (e.g., human CD 3) and the second target binding domain specifically binds to CD28 (e.g., human CD 28). In some embodiments, the first target binding domain specifically binds to CD28 (e.g., human CD 28) and the second target binding domain specifically binds to CD3 (e.g., human CD 3).

In some embodiments of these single chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain.

In some embodiments of these single chain chimeric polypeptides, the soluble tissue factor domain and the second target binding domain are directly adjacent to each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the second target binding domain.

In some embodiments of these single-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of these single-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each an antigen binding domain (e.g., any of the exemplary antigen binding domains described herein). In some embodiments of these single chain chimeric polypeptides, the antigen binding domain comprises an scFv or a single domain antibody.

Non-limiting examples of scFv that specifically bind to CD3 can include sequences that are at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS(SEQ ID NO:151)。

in some embodiments, an scFv that specifically binds to CD3 may be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC(SEQ ID NO:152)。

non-limiting examples of scFv that specifically bind to CD28 can include sequences that are at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(SEQ ID NO:153)。

in some embodiments, an scFv that specifically binds to CD28 may be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG (SEQ ID NO: 154).

In some embodiments of these single-chain chimeric polypeptides, the first target binding domain and/or the second target binding domain is a soluble receptor (e.g., a soluble CD28 receptor or a soluble CD3 receptor). In some embodiments of these single chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(SEQ ID NO:155)。

in some embodiments, the single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG(SEQ ID NO:156)。

in some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR(SEQ ID NO:157)。

In some embodiments, the single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCCCAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG(SEQ ID NO:158)。

exemplary embodiments of type B Single chain chimeric Polypeptides

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and/or the second target binding domain can independently specifically bind to an IL-2 receptor (e.g., a human IL-2 receptor).

In some embodiments of these single chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain.

In some embodiments of these single chain chimeric polypeptides, the soluble tissue factor domain and the second target binding domain are directly adjacent to each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the second target binding domain.

In some embodiments of these single chain chimeric polypeptides, the first target binding domain and the second target binding domain are soluble human IL-2 proteins. Non-limiting examples of IL-2 proteins that specifically bind to the IL-2 receptor may include sequences that are at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:78)。

in some embodiments, an IL-2 protein that specifically binds to an IL-2 receptor may be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT(SEQ ID NO:159)。

in some embodiments, an IL-2 protein that specifically binds to an IL-2 receptor may be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACC(SEQ ID NO:160)。

In some embodiments of these single chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:161)。

in some embodiments, the single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT(SEQ ID NO:162)。

in some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:163)。

In some embodiments, the single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT(SEQ ID NO:164)。

exemplary embodiments of the C-type Single-chain chimeric polypeptide

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and/or the second target binding domain can independently specifically bind to an IL-15 receptor (e.g., a human IL-15 receptor).

In some embodiments of these single chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain.

In some embodiments of these single chain chimeric polypeptides, the soluble tissue factor domain and the second target binding domain are directly adjacent to each other. In some embodiments of these single-chain chimeric polypeptides, the single-chain chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the second target binding domain.

In some embodiments of these single chain chimeric polypeptides, the first target binding domain and the second target binding domain are soluble human IL-15 proteins. Non-limiting examples of IL-15 proteins that specifically bind to the IL-15 receptor may include sequences that are at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:82)。

in some embodiments, an IL-15 protein that specifically binds to an IL-15 receptor may be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACC(SEQ ID NO:165)。

in some embodiments, an IL-15 protein that specifically binds to an IL-15 receptor may be encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:166)。

In some embodiments of these single chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein.

In some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:167)。

in some embodiments, the single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:168)。

in some embodiments, a single-chain chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:169)。

In some embodiments, the single-chain chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCAACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:170)。

exemplary type A multiple chain chimeric polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-18 or a receptor for IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises an scFv or single domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to a receptor for IL-18 or IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-12, and the second target binding domain specifically binds to a receptor for IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-18, and the second target binding domain specifically binds to a receptor for IL-12.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-18 (e.g., soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-18 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:109)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT(SEQ ID NO:171)。

In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises soluble IL-12 (e.g., soluble human IL-12). In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-15 includes the sequence of soluble human IL-12β (p 40) and the sequence of soluble human IL-12α (p 35). In some embodiments of these multi-chain chimeric polypeptides, soluble IL-15 human IL-15 also includes a linker sequence (e.g., any of the exemplary linker sequences described herein) between the sequence of soluble IL-12β (p 40) and the sequence of soluble human IL-12α (p 35). In some embodiments of these multi-chain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-12 β (p 40) comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:81)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-12 β (p 40) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC(SEQ ID NO:172)。

In some embodiments of these multiple chain chimeric polypeptides, soluble human IL-12α (p 35) includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:80)。

in some embodiments of these multiple chain chimeric polypeptides, soluble human IL-12α (p 35) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC(SEQ ID NO:173)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:174)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 175).

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:176)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGCTACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:177)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:178)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:179)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:180)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:181)。

exemplary type B multiple chain chimeric polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-21 or TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble TGF- β receptor (e.g., a soluble TGFR βrii receptor). In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-21 or soluble TGF- β receptor (e.g., soluble TGFR βrii receptor). In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the receptor for IL-21 or TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-21 and the second target binding domain specifically binds to TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds TGF- β, and the second target binding domain specifically binds to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-21 (e.g., soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:83)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

in some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises a soluble TGF- β receptor (e.g., a soluble TGFR βrii receptor (e.g., a soluble human TGFR βrii receptor)). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

in some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (SEQ ID NO: 186).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

In some embodiments of these multi-chain chimeric polypeptides, the human tgfbetarii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:189)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:190)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ IDNO:191)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:192)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:193)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA(SEQ ID NO:194)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:195)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA(SEQ ID NO:196)。

exemplary C-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7 or a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-21 or soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the receptor for IL-21 or IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-21, and the second target binding domain specifically binds to a receptor for IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-7, and the second target binding domain specifically binds to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-21 (e.g., soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANT GNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:83)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC(SEQ ID NO:197)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO:79)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC(SEQ ID NO:198)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:199)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:200)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MGVKVLFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ IDNO:201)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:202)

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:203)

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA(SEQ ID NO:204)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MGVKVLFALICIAVAEADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:205)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA(SEQ ID NO:206)。

exemplary D-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7 or a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-21 or soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the receptor for IL-21 or IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-21, and the second target binding domain specifically binds to a receptor for IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-7, and the second target binding domain specifically binds to a receptor for IL-21.

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:83)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC(SEQ ID NO:197)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

in some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO:79)。

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC(SEQ ID NO:198)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:207)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:208)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:209)。

In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:210)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:211)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:212)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:213)。

In some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:214)。

exemplary E-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-18 (e.g., soluble human IL-18), a receptor for IL-12 (e.g., soluble human IL-12), or CD16 (e.g., anti-CD 16 scFv). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a receptor for CD16 or IL-12.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional antigen binding domain is an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain, the second target binding domain, and the additional antigen binding domain are each an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises an scFv or single domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to a receptor for IL-18 or IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-12, and the second target binding domain specifically binds to a receptor for IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-18, and the second target binding domain specifically binds to a receptor for IL-12. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to CD16 and the second target binding domain specifically binds to a receptor for IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-18, and the second target binding domain specifically binds to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-18 (e.g., soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-18 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:109)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-18 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT(SEQ ID NO:171)。

in some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises soluble IL-12 (e.g., soluble human IL-12). In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-15 includes the sequence of soluble human IL-12β (p 40) and the sequence of soluble human IL-12α (p 35). In some embodiments of these multi-chain chimeric polypeptides, soluble IL-15 (e.g., soluble human IL-15) also includes a linker sequence (e.g., any of the exemplary linker sequences described herein) between the sequence of soluble IL-12β (p 40) and the sequence of soluble human IL-12α (p 35). In some embodiments of these multi-chain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-12 β (p 40) comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS(SEQ ID NO:81)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-12 β (p 40) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC(SEQ ID NO:172)。

in some embodiments of these multiple chain chimeric polypeptides, soluble human IL-12α (p 35) includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS(SEQ ID NO:80)。

In some embodiments of these multiple chain chimeric polypeptides, soluble human IL-12α (p 35) is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC(SEQ ID NO:173)。

in some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain comprises an scFv that specifically binds to CD16 (e.g., an anti-CD 16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a light chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH(SEQ ID NO:215)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT(SEQ ID NO:216)。

In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a heavy chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:217)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO:218)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 174).

In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:175)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:176)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGCTACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:177)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:223)。

In some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO:224)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:225)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO:226)。

exemplary type F multiple chain chimeric polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7 (e.g., soluble human IL-7), CD16 (e.g., anti-CD 16 scFv), or IL-21 (e.g., soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a receptor for CD16 or IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional antigen binding domain is an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain, the second target binding domain, and the additional antigen binding domain are each an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises an scFv or single domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain specifically binds to the receptor IL-7, and the second target binding domain specifically binds to the receptor for CD16 or IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain comprises a soluble IL-7 protein. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-7 protein is soluble human IL-7. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen binding domain comprises a target binding domain that specifically binds to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain comprises an scFv that specifically binds to CD 16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain specifically binds to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to CD 16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-7 (e.g., soluble human IL-7).

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO:79)。

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC(SEQ ID NO:198)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT(SEQ ID NO:227)。

in some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:83)。

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC(SEQ ID NO:197)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

in some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain comprises an scFv that specifically binds to CD16 (e.g., an anti-CD 16 scFv).

In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a light chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH(SEQ ID NO:215)。

In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT(SEQ ID NO:216)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a heavy chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:217)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO:218)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:207)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:208)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:209)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:210)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:232)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:233)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:234)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:235)。

Exemplary G-type Multi-chain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor of tgfβ (e.g., human tgfβrii receptor), CD16 (e.g., anti-CD 16 scFv), or IL-21 (e.g., soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a receptor for CD16 or IL-21.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional antigen binding domain is an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain, the second target binding domain, and the additional antigen binding domain are each an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises an scFv or single domain antibody.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for TGF- β, CD16, or IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain specifically binds to TGF- β and the second target binding domain specifically binds to a receptor for CD16 or IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain is a soluble TGF- β receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble TGF- β receptor is a soluble tgfbetarii receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain comprises an antigen-binding domain that specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen binding domain comprises an scFv that specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain specifically binds to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain comprises soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to a receptor for IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to CD16.

In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises a tgfbetarii receptor (e.g., a soluble human tgfbetarii receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

in some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:188)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble human tgfbetarii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

In some embodiments of these multi-chain chimeric polypeptides, the sequence of soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:83)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC(SEQ ID NO:197)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a light chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH(SEQ ID NO:215)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT(SEQ ID NO:216)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a heavy chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:217)。

In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 218).

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:236)。

In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:237)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:239)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:232)。

In some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:233)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:234)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:235)。

exemplary H-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise soluble IL-7 (e.g., soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO:79)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT(SEQ ID NO:227)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:207)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:208)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:209)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:210)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:203)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA (SEQ ID NO: 204).

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:250)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:251)。

exemplary type I multiple chain chimeric polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain each independently specifically bind to TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are soluble TGF- β receptors (e.g., soluble tgfbetarii receptors, e.g., soluble human tgfbetarii). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

in some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:188)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:236)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:237)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:239)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:193)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:257)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:195)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:259)。

Exemplary J-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7, a receptor for IL-21, or a receptor for CD 137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a receptor for IL-21 (e.g., soluble IL-21, e.g., soluble human IL-21) or a receptor for CD137L (e.g., soluble CD137L, e.g., soluble human CD 137L).

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments, the second chimeric polypeptide may include an additional target binding domain. In some embodiments, additional target binding domains and

in some embodiments of these multi-chain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional target binding domain is an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain, the second target binding domain, and the additional target binding domain are each an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises an scFv or single domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-7, and the second target binding domain specifically binds to a receptor for IL-21 or a receptor for CD 137L. In some embodiments, the additional target binding domain specifically binds to a receptor for IL-21 or a receptor for CD 137L.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is soluble IL-7 (e.g., soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO:79)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT(SEQ ID NO:227)。

in some embodiments of these multi-chain chimeric polypeptides, the second target binding domain or additional target binding domain specifically binds to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain or additional target binding domain is soluble IL-21 (e.g., soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:83)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

in some embodiments of these multi-chain chimeric polypeptides, the second target binding domain specifically binds to a receptor for CD 137L. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to a receptor for CD 137L. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain and/or additional target binding domain is soluble CD137L (e.g., soluble human CD 137L).

In some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:260)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA(SEQ ID NO:261)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO:262)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO:263)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:207)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:208)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:209)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:210)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:268)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA(SEQ ID NO:269)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:270)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA(SEQ ID NO:271)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO:272)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO:273)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO:274)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO:275)。

Exemplary K-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7 or TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to a receptor for IL-7 and the second target binding domain specifically binds to TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF-beta and the second target binding domain specifically binds to a receptor for IL-7.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises a soluble IL-7 protein (e.g., a soluble human IL-7 protein). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 protein comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH(SEQ ID NO:79)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human IL-7 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT(SEQ ID NO:227)。

in some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises a target binding domain that specifically binds to TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain is a soluble TGF- β receptor (e.g., a soluble tgfbetarii receptor, such as a soluble human tgfbetarii receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

in some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:188)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:207)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:208)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:209)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:210)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:193)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:257)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:195)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:259)。

Exemplary L-shaped Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF- β, a receptor for IL-21, or a receptor for CD 137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a receptor for IL-21 (e.g., soluble IL-21, e.g., soluble human IL-21) or a receptor for CD137L (e.g., soluble CD137L, e.g., soluble human CD 137L).

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional target binding domain is an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain, the second target binding domain, and the additional target binding domain are each an agonistic antigen binding domain. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises an scFv or single domain antibody.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF- β, and the second target binding domain specifically binds to the receptor for IL-21 or the receptor for CD 137L.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is a soluble TGF- β receptor (e.g., a soluble tgfbetarii receptor, such as a soluble human tgfbetarii receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 184).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 188).

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain or additional target binding domain specifically binds to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain or additional target binding domain comprises soluble IL-21 (e.g., soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83).

In some embodiments of these multi-chain chimeric polypeptides, soluble human IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain or additional target binding domain specifically binds to a receptor of CD 137L. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain and/or additional target binding domain comprises soluble CD137L (e.g., soluble human CD 137L).

In some embodiments of these multi-chain chimeric polypeptides, soluble CD137L comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:260)。

in some embodiments of these multi-chain chimeric polypeptides, soluble CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 261).

In some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L comprises a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO:262)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO:263)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:236)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:237)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:239)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:268)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA(SEQ ID NO:269)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:270)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA(SEQ ID NO:271)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO:272)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO:273)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO:274)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO:275)。

exemplary M-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for TGF- β or IL-21. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a receptor for IL-21 (e.g., soluble IL-21, e.g., soluble human IL-21) or TGF-beta (e.g., soluble TGF-beta receptor, e.g., soluble TGF-beta RII receptor).

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF-beta and the second target binding domain specifically binds to a receptor for TGF-beta or IL-21. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is a soluble TGF- β receptor (e.g., a soluble tgfbetarii receptor, such as a soluble human tgfbetarii receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

in some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:188)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

in some embodiments of these multi-chain chimeric polypeptides, the second target binding domain specifically binds to a receptor for IL-21. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises soluble IL-21 (e.g., human soluble IL-21). In some embodiments of these multi-chain chimeric polypeptides, soluble IL-21 comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:83)。

In some embodiments of these multi-chain chimeric polypeptides, soluble IL-21 is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:182)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:236)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:237)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:239)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ IDNO:300)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:301)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS(SEQ ID NO:302)。

In some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC(SEQ ID NO:303)。

exemplary N-type multiple chain chimeric polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF- β or CD16. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to CD16 (e.g., anti-CD 16 scFv) or TGF- β (e.g., soluble TGF- β receptor, e.g., soluble tgfbetarii receptor).

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF- β, and the second target binding domain specifically binds to TGF- β or CD16. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is a soluble TGF- β receptor (e.g., a soluble tgfbetarii receptor, such as a soluble human tgfbetarii receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

in some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:188)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain specifically binds to CD16. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises an anti-CD 16 scFv. In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a light chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH(SEQ ID NO:215)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a light chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT(SEQ ID NO:216)。

in some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 comprises a heavy chain variable domain comprising a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:217)。

In some embodiments of these multi-chain chimeric polypeptides, the scFv that specifically binds to CD16 is encoded by a heavy chain variable domain sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO:218)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:236)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:237)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:239)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:308)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO:309)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR(SEQ ID NO:310)。

In some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG(SEQ ID NO:311)。

exemplary O-type Multichain chimeric Polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a receptor for TGF- β or CD137L. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a receptor for TGF- β (e.g., a soluble TGF- β receptor, e.g., a soluble tgfbetarii receptor) or CD137L.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF- β and the second target binding domain specifically binds to CD137L. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain or additional target binding domain is a soluble TGF- β receptor (e.g., a soluble tgfbetarii receptor, such as a soluble human tgfbetarii receptor).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:188)。

In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

in some embodiments these multi-chain chimeric polypeptides, the second target binding domain comprises a soluble CD137L protein (e.g., a soluble human CD137L protein). In some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L comprises a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:260)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to: CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 261).

In some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L comprises a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI(SEQ ID NO:262)。

in some embodiments of these multi-chain chimeric polypeptides, soluble human CD137L is encoded by a sequence that is at least 80% identical (e.g., at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC(SEQ ID NO:263)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: 3PPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 236).

In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC(SEQ ID NO:237)。

in some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 239).

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:316)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA(SEQ ID NO:317)。

in some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE(SEQ ID NO:318)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 319).

Exemplary P-type multiple chain chimeric polypeptides

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each specifically bind to TGF- β. In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. Non-limiting examples of this type of multi-chain chimeric polypeptide are shown in figures 209 and 210.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain in the pair of affinity domains in the first chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain in a pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain and the second target binding domain in a pair of affinity domains in the second chimeric polypeptide.

In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of a pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain in the second chimeric polypeptide.

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain may be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary affinity domains described herein.

In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF- β and the second target binding domain specifically binds to TGF- β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and/or the second target binding domain is a soluble TGF- β receptor (e.g., a soluble tgfbetarii receptor, such as a soluble human tgfbetarii receptor).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a first sequence of soluble human TGFR βrii and a second sequence of soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFR βrii comprises a linker disposed between a first sequence of the soluble human TGFR βrii and a second sequence of the soluble human TGFR βrii. In some embodiments of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:183)。

in some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:184)。

in some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:185)。

In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFR βrii receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:186)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor comprises a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ ID NO:188)。

in some embodiments of these multi-chain chimeric polypeptides, the soluble TGF- β receptor is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:187)。

In some embodiments, the first chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

in some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAAAACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT(SEQ ID NO:239)。

in some embodiments, the first chimeric polypeptide can include a soluble IL-15 comprising a D8N amino acid substitution and have a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

(Signal peptide)

MGVKVLFALICIAVAEA

(Single chain human TGF-beta receptor II homodimer)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(tissue factor)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(IL-15D8N)

NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:238)。

In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC (Single chain human TGF-beta receptor II homodimer)

ATCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA

(human IL-15D 8N)

AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT(SEQ ID NO:244)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:240)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:241)。

In some embodiments, the second chimeric polypeptide may comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:242)。

in some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the sequence:

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG(SEQ ID NO:243)。

methods of treating aging-related diseases or disorders

Provided herein are methods of treating an aging-related disease or disorder (e.g., any of the exemplary types of aging-related diseases or disorders described herein or known in the art) in a subject in need thereof, comprising administering a therapeutically effective amount of one or more Natural Killer (NK) cell activators (e.g., any of the Natural Killer (NK) cell activators described herein or known in the art) to a subject identified as having an aging-related disease or disorder (e.g., any of the exemplary types of aging-related diseases or disorders described herein or known in the art).

Provided herein are methods of treating an aging-related disease or disorder (e.g., any of the exemplary types of aging-related diseases or disorders described herein or known in the art) in a subject in need thereof, comprising administering a therapeutically effective amount of activated NK cells (e.g., any of the activated NK cells described herein or known in the art) to a subject identified as having an aging-related disease or disorder (e.g., any of the exemplary types of aging-related diseases or disorders described herein or known in the art).

Some embodiments of these methods further comprise: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are haploid NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are allogeneic resting NK cells. In some embodiments of these methods, the resting NK cells are artificial NK cells. In some embodiments of any of these methods, the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

In some embodiments of these methods, the liquid medium is a serum-free liquid medium. In some embodiments of any one of the methods described herein, the liquid medium is a chemically-defined liquid medium. Some embodiments of these methods further comprise isolating the activated NK cells (and optionally further administering a therapeutically effective amount of the activated NK cells to a subject, such as any of the subjects described herein). In some embodiments of these methods, the contacting step is performed for a period of time ranging from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of any one of the methods described herein, the aging-related disease or disorder is selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, skin diseases, premature senility diseases and fragile diseases.

Non-limiting examples of cancers include: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

A non-limiting example of an autoimmune disease is type 1 diabetes.

Non-limiting examples of metabolic diseases include: obesity, lipodystrophy and type 2 diabetes.

Non-limiting examples of neurodegenerative diseases include: alzheimer's disease, parkinson's disease and dementia.

Non-limiting examples of cardiovascular diseases include: coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

Non-limiting examples of dermatological disorders include: wound healing, alopecia, wrinkles, senile lentigo, thinning of skin, xeroderma pigmentosum and congenital dysplastic keratosis.

Non-limiting examples of premature senility diseases include: premature senility and Huzison-Ji Erfu premature senility syndrome.

Non-limiting examples of fragile diseases include: friability, vaccination response, osteoporosis and sarcopenia.

In some embodiments of any one of the aging-related diseases or disorders described herein, the aging-related disease or disorder is selected from the group consisting of: age-related macular degeneration, osteoarthritis, lipoatrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related renal dysfunction, and chemically induced renal dysfunction.

In some embodiments of any of the aging-related diseases or disorders described herein, the aging-related disease or disorder is type 2 diabetes or atherosclerosis.

In some embodiments of any one of the methods described herein, the subject has been diagnosed with or identified as having an aging-related disease or disorder (e.g., any one of the exemplary aging-related diseases or disorders described herein). Some embodiments of any of the methods described herein can include the step of selecting a subject identified or diagnosed with an aging-related disease or disorder (e.g., any of the exemplary aging-related diseases or disorders described herein).

In some embodiments of these methods, for example, administration results in a decrease in the number of senescent cells in a subject's target tissue compared to the number of senescent cells in the subject's target tissue prior to treatment (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 99%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 99%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 99%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 20% to about 45%, about 40%, about 25% to about 35%, about 25% to about 95%, about 25% of the like about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 25% to about 30%, about 30% to about 99%, about 30% to about 95%, about 30% to about 90%, about 30% to about 85%, about 30% to about 80%, about 30% to about 75%, about 30% to about 70%, about 30% to about 65%, about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 99%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 30% to about 70%, about 30% to about 65%, about 30% to about 55%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35% of the like, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 99%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 45% to about 60%, about 45% to about 45%, about 45% and about 45% of the like. About 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 99%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 99%, about 50% to about 60%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 55% and about 80%, about 55% to about 80%, about 50% to about 55% to about 80%, about 55% to about 55% of the like, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75%, about 75% to about 70%, about 60% to about 65%, about 65% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 99%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about 99% to about 85%, or about 95%.

In some embodiments of these methods, administration increases (e.g., by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 99%, or by about 10% to about 500% (or any subrange of this range described herein)) the level of IFN- γ, cytotoxic granzyme and/or perforin in the subject compared to the level in a pre-treatment subject or a similar control subject that has not received the treatment.

In some embodiments, the methods are capable of reducing the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., as compared to the number, severity, or frequency of one or more symptoms of cancer in a subject prior to treatment). In some embodiments, the methods can result in a decrease in the volume of one or more solid tumors in a subject (e.g., about 1% to about 99%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 99%, about 5% to about 95%, about 5% to about 90%, about 5% to about 85%, about about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65%, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 99%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80%, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about, about 10% to about 55%, about 10% to about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 99%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 30%, about 15% to about 30%, about about 20% to about 99%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, and about about 20% to about 35% reduction, about 20% to about 30% reduction, about 20% to about 25% reduction, about 25% to about 99% reduction, about 25% to about 95% reduction, about 25% to about 90% reduction, about 25% to about 85% reduction, about 25% to about 80% reduction, about 25% to about 75% reduction, about 25% to about 70% reduction, about 25% to about 65% reduction, about 25% to about 60% reduction, about 25% to about 55% reduction, about 80% reduction, about, about 25% to about 50% reduction, about 25% to about 45% reduction, about 25% to about 40% reduction, about 25% to about 35% reduction, about 25% to about 30% reduction, about 30% to about 99% reduction, about 30% to about 95% reduction, about 30% to about 90% reduction, about 30% to about 85% reduction, about 30% to about 80% reduction, about 30% to about 75% reduction, about 30% to about 70% reduction, about 30% to about 65% reduction, about light-emitting device, and method of making the same about 30% to about 60%, about 30% to about 55%, about 30% to about 50%, about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 99%, about 35% to about 95%, about 35% to about 90%, about 35% to about 85%, about 35% to about 80%, about 35% to about 75%, about 35% to about 70%, about about 35% to about 65%, about 35% to about 60%, about 35% to about 55%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 99%, about 40% to about 95%, about 40% to about 90%, about 40% to about 85%, about 40% to about 80%, about 40% to about 75%, about 40% to about 70%, about 40% to about 65%, about 40% to about 60%, about 40% to about 55%, about 40% to about 50%, about 40% to about 45%, about 45% to about 99%, about 45% to about 95%, about 45% to about 90%, about 45% to about 85%, about 45% to about 80%, about 45% to about 75%, about 45% to about 70%, about 45% to about 65%, about% > About 45% to about 60%, about 45% to about 55%, about 45% to about 50%, about 50% to about 99%, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70%, about 50% to about 65%, about 50% to about 60%, about 50% to about 55%, about 55% to about 99%, about 55% to about 95%, about 55% to about 90%, about 55% to about 85%, about 55% to about 80%, about 55% to about 75%, about 55% to about 70%, about 55% to about 65%, about 55% to about 60%, about 60% to about 99%, about 60% to about 95%, about 60% to about 90%, about 60% to about 85%, about 55% to about 75%, about 55% to about 70%, about 55% to about 60%, about 60% to about 85%, about about 60% to about 80%, about 60% to about 75%, about 60% to about 70%, about 60% to about 65%, about 65% to about 99%, about 65% to about 95%, about 65% to about 90%, about 65% to about 85%, about 65% to about 80%, about 65% to about 75%, about 65% to about 70%, about 70% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, about 70% to about 75%, about 75% to about 99%, about 75% to about 95%, about 75% to about 90%, about 75% to about 85%, about 75% to about 80%, about 80% to about 99%, about 80% to about 95%, about 80% to about 90%, about 80% to about 85%, about, about 85% to about 99% reduced, about 85% to about 95% reduced, about 85% to about 90% reduced, about 90% to about 99% reduced, about 90% to about 95% reduced, or about 95% to about 99% reduced) (e.g., as compared to the volume of one or more solid tumors prior to or at the beginning of treatment). In some embodiments, the methods can reduce (e.g., reduce by about 1% to about 99%, or any subrange of this range described herein) the risk of metastasis or the occurrence of one or more additional metastases in a subject (e.g., as compared to the risk of metastasis or the occurrence of one or more additional metastases in a subject prior to treatment or in a similar subject or population of subjects administered different treatments).

In some embodiments, the methods may result in the treatment of a metabolic disorder in a subject. In some embodiments, the treatment of the metabolic disease can result in, for example, one or more (e.g., two, three, four, five, or six) of: improved glucose tolerance, improved glucose utilization, reduced severity or exacerbation of diabetic osteoarthritis, reduced severity or exacerbation of dermatological lesions, reduced severity or exacerbation of ketosis, reduced production of autoantibodies to islet cells, increased insulin sensitivity, reduced mass, and reduced body mass index. The subject's response to treatment can be monitored by measuring fasting glucose or glucose tolerance according to standard techniques. Typically, the blood glucose is reduced according to the method so as to achieve a blood glucose level characterized by a fasting blood glucose of less than 100mg/dL or a two hour 75g oral glucose tolerance test value of less than 140mg/dL. In some embodiments, the response to treatment may include determining other factors associated with pre-diabetes, new onset diabetes, or active diabetes, including blood pressure, body mass index, PPAR-gamma function, lipid metabolism, glycosylated hemoglobin (H1 c), and renal function.

In some embodiments, these methods can eliminate or reduce the risk of neurodegenerative diseases, reduce their severity or delay their onset, including biochemical, histological and/or behavioral symptoms of the disease, complications thereof, and intermediate pathological phenotypes that occur during exacerbation of the disease.

In some embodiments, effective treatment of skin disorders can be assessed by any method described herein or known in the art, including examining skin conditions, including skin color, humidity, temperature, texture, mobility, and swelling, as well as skin lesions (as compared to skin conditions prior to treatment).

In some embodiments, effective treatment of autoimmune disease can be assessed by any of the methods described herein or known in the art, including monitoring whole blood count analysis of freshly isolated PBMCs, analysis of total Ig levels, and serum autoantibody titers.

In some embodiments, effective treatment of fragile diseases can be assessed by any of the methods described herein or known in the art, including monitoring bone mineral density, bone structure and geometry, biomedical markers of bone turnover, vitamin D measurements, canola behavior state (Karnofsky performance status) and ECOG score, and vaccination response.

Methods of killing or reducing the number of senescent cells in a subject

Provided herein are methods of killing or reducing the number of senescent cells (e.g., senescent cells of any of the exemplary types described herein or known in the art) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more NK cell activators (e.g., any of the NK cell activators described herein or known in the art).

Also provided herein are methods of killing or reducing the number of senescent cells (e.g., any of the exemplary types of senescent cells described herein or known in the art) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of activated NK cells (e.g., any of the activated NK cells described herein or known in the art).

Some embodiments of these methods further comprise: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are haploid NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are allogeneic resting NK cells. In some embodiments of these methods, the resting NK cells are artificial NK cells. In some embodiments of any of these methods, the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

In some embodiments of these methods, the liquid medium is a serum-free liquid medium. In some embodiments of any one of the methods described herein, the liquid medium is a chemically-defined liquid medium. Some embodiments of these methods further comprise isolating the activated NK cells (and further administering a therapeutically effective amount of the activated NK cells to a subject, such as any of the subjects described herein). In some embodiments of these methods, the contacting step is performed for a period of time ranging from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of these methods, the senescent cell is a senescent cancer cell, a senescent monocyte, a senescent lymphocyte, a senescent astrocyte, a senescent microglial cell, a senescent neuron, a senescent tissue fibroblast, a senescent dermal fibroblast, a senescent keratinocyte, or other differentiated tissue-specific dividing functional cell. In some embodiments of these methods, the senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced senescent cells.

In some embodiments of these methods, the subject has been identified or diagnosed with an aging-related disease or disorder (e.g., any of the aging-related diseases or disorders described herein or known in the art). In some embodiments of any one of the aging-related diseases or disorders described herein, the aging-related disease or disorder is selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, skin diseases, premature aging and fragile diseases.

Non-limiting examples of cancers include: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

A non-limiting example of an autoimmune disease is type 1 diabetes.

Non-limiting examples of metabolic diseases include: obesity, lipodystrophy and type 2 diabetes.

Non-limiting examples of neurodegenerative diseases include: alzheimer's disease, parkinson's disease and dementia.

Non-limiting examples of cardiovascular diseases include: coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

Non-limiting examples of dermatological disorders include: wound healing, alopecia, wrinkles, senile lentigo, thinning of skin, xeroderma pigmentosum and congenital dysplastic keratosis.

Non-limiting examples of premature senility diseases include: premature senility and Huzison-Ji Erfu premature senility syndrome.

Non-limiting examples of fragile diseases include: friability, vaccination response, osteoporosis and sarcopenia.

In some embodiments of any one of the aging-related diseases or disorders described herein, the aging-related disease or disorder is selected from the group consisting of: age-related macular degeneration, osteoarthritis, lipoatrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related renal dysfunction, and chemically induced renal dysfunction.

In some embodiments of any of the aging-related diseases or disorders described herein, the aging-related disease or disorder is type 2 diabetes or atherosclerosis.

In some embodiments of these methods, for example, administration results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 10% to about 99% (or any subrange of this range described herein)) in the target tissue in the subject compared to the number of senescent cells in the target tissue in the subject prior to treatment. In some embodiments of these methods, the target tissue in the subject may be one or more of the following: adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature, bone tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

In some embodiments of these methods, administration increases (e.g., by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 99%, or by about 10% to about 500% (or any subrange of this range described herein)) the level of IFN- γ, cytotoxic granzyme and/or perforin in the subject compared to the level in a pre-treatment subject or a similar control subject that has not received the treatment.

In some embodiments of these methods, the number of senescent cells in a target tissue (e.g., any of the target tissues described herein) can be determined by immunostaining a biopsy sample. In some embodiments of these methods, the number of senescent cells in a target tissue (e.g., any of the target tissues described herein) can be observed indirectly through improvement of one or more conditions (e.g., any of the conditions of the aging-related diseases or conditions described herein) of the subject.

Senescent cells

Senescent cells exhibit important and unique characteristics including changes in morphology, chromatin organization, gene expression, and metabolism. There are several biochemical and functional properties associated with cellular aging, such as (i) inhibitors of cyclin-dependent kinases, p16 ^INK4a And p21 ^CIP1 (ii) the presence of a marker of senescence-associated beta-galactosidase, i.e. lysosomal activity, (iii) the occurrence of down-regulation of senescence-associated heterochromatin foci and lamin B1 levels, (iv) resistance to apoptosis caused by increased expression of anti-apoptotic BCL family proteins, and (v) CD26 (DPP 4), CD36 (Scavenger) receptor, fork-box 4 (FOXO 4) and secretion Carrier membrane protein 4 (SCAMP 4) was up-regulated. Senescent cells also express an inflammatory feature, the so-called senescence-associated secretory phenotype (SASP). By SASP, senescent cells produce a wide range of inflammatory cytokines (IL-6, IL-8), growth factors (TGF-beta), chemokines (CCL-2), and matrix metalloproteinases (MMP-3, MMP-9), which operate in a cell-autonomous manner to enhance senescence (autocrine effects), and communicate with and alter the microenvironment (paracrine effects). SASP factors may promote tumor suppression by triggering senescence monitoring, i.e., immune-mediated senescent cell clearance. However, chronic inflammation is also a known driver of tumorigenesis, and there is increasing evidence that chronic SASP can also enhance cancer metastasis and aging-related diseases.

The secretory profile of senescent cells depends on the circumstances. For example, mitochondrial dysfunction-related aging (MiDAS) induced by different mitochondrial dysfunctions in human fibroblasts causes the appearance of SASP lacking IL-1 dependent inflammatory factors. The decrease in the NAD+/NADH ratio activates AMPK signaling, which induces MiDAS by activating p 53. As a result, p53 inhibits NF- κb signaling, a key inducer of pro-inflammatory SASP. In contrast, cellular senescence caused by sustained DNA damage in human cells induces inflammatory SASPs that rely on the activation of Ataxia Telangiectasia Mutated (ATM) kinase, and not on the activation of p 53. In particular, the expression and secretion levels of IL-6 and IL-8 are increased. Also revealed the ectopic expression of p16 ^INK4a And p21 ^CIP1 The resulting cellular senescence induced the senescent phenotype of human fibroblasts, and the absence of inflammatory SASP, indicated that growth arrest itself did not stimulate SASP.

One of the most important features of aging is stable growth arrest. This is through two important paths, p16 ^INK4 a/Rb and p53/p21 ^CIP1 Both of these pathways are critical for tumor suppression. DNA damage causes: (1) High deposition of γh2ax (histone encoding gene) and 53BP1 (involved in DNA damage response) in chromatin: this causes activation of the kinase cascade, ultimately leading to P53 activation, and (2) activation of p16.sup.INK4a and ARF (both encoded by CDKN 2A) and p15.sup.INK4b (encoded by CDKN 2B): p 53-induced cyclin-dependent kinase inhibitors (p 21) ^CIP1 ) And blocks genes (CDK 4 and CDK 6) of cell cycle progression together with both p16.sup.INk4a and p15.sup.INk4b. This ultimately causes insufficient phosphorylation of retinoblastoma protein (Rb) and arrest of the cell cycle in the G1 phase.

Selective killing of senescent cells has been shown to significantly improve the healthy life of mice under normal aging conditions, and to improve the outcome of age-related disease or cancer therapy (Ovadea, J Clin invest.128 (4): 1247-1254, 2018). In nature, senescent cells are often removed by innate immune cells. The induction of senescence not only prevents the potential proliferation and transformation of damaged/altered cells, but also promotes tissue repair by producing SASP factors that act primarily as chemoattractants for Natural Killer (NK) cells (e.g., IL-15 and CCL 2) and chemoattractants for macrophages (e.g., CFS-1 and CCL 2). These innate immune cells mediate immune surveillance mechanisms that eliminate stressed cells. Senescent cells generally up-regulate NK cell activating receptors NKG2D and DNAM1 ligands, which belong to the stress-inducible ligand family: important components of first-line immunity against infectious diseases and malignancies. Upon receptor activation, NK cells can then specifically induce death of senescent cells through their cytolytic machinery. The role of NK cells in the immunological monitoring of senescent cells has been pointed out in liver fibrosis (Sagiv, oncogene 32 (15): 1971-1977, 2013), hepatocellular carcinoma (Iannello, J Exp Med 210 (10): 2057-2069, 2013), multiple myeloma (Soriani, blood 113 (15): 3503-3511, 2009) and glioma cells stressed by mevalonate pathway dysfunctions (Ciaglia, int J Cancer 142 (1): 176-190, 2018). Endometrial cells undergo acute cellular senescence and do not differentiate into decidua cells. Differentiated decidua cells secrete IL-15, thereby recruiting uterine NK cells to target and eliminate undifferentiated senescent cells, thus helping to remodel and rejuvenate the endometrium (Brighton, elife 6:e31274, 2017). In a similar mechanism, senescent liver satellite cells expressing p53 bias polarization of resident Kupfer (Kupfer) macrophages and newly infiltrated macrophages towards the pro-inflammatory M1 phenotype during liver fibrosis, which shows senescent cell lysis activity (senolytic activity). F4/80+ macrophages have been shown to play a key role in the clearance of mouse uterine senescent cells to maintain postpartum uterine function.

Senescent cells recruit NK cells primarily through up-regulation of ligands for NKG2D (expressed on NK cells), chemokines and other SASP factors. In vivo models of liver fibrosis have shown that activated NK cells are effective in eliminating senescent cells (Krizhanovsky, cell 134 (4): 657-667, 2008). Studies a variety of models have been described to study aging, including liver fibrosis (Krizhanovsky, cell 134 (4): 657-667, 2008), osteoarthritis (Xu, J Gerontol A Biol Sci Med Sci (6): 780-785, 2017) and parkinson's disease (Chinta, cell Rep 22 (4): 930-940, 2018). Animal models for studying senescent cells are described in: krizhanovsky, cell 134 (4): 657-667, 2008; baker, nature 479 (7372): 232-236, 2011; farr, nat Med 23 (9): 1072-1079,2017; bourgeois, FEBS Lett 592 (12): 2083-2097,2018; xu, nat Med 24 (8): 1246-1256, 2018).

Senescence is a form of irreversible growth arrest, accompanied by phenotypic changes, resistance to apoptosis, and activation of injury-aware signaling pathways. Cell senescence was originally described in cultured human fibroblasts, which lost their proliferative capacity, reaching permanent arrest after about 50 population doublings (known as the Hayflick Limit (Hayflick et al, exp. Cell Res.25:585-621, 1961.) He observed a "replicative senescence" phenomenon in cultures of non-immortalized human fibroblasts, caused by progressive shortening of telomeres at each Cell division, representing a physiological response preventing genomic instability and accumulation of DNA damage (He et al, cell 169 (6): 1000-1011, 2017).

Aging is thought to be a stress response that can be induced by a wide range of intrinsic and extrinsic injuries, including oxidative and genotoxic stress, DNA damage, telomere loss, or oncogene activation, mitochondrial dysfunction, or chemotherapeutics (McHugh et al, j.cell biol.217 (1): 65-77, 2018). This accelerated senescence response, which is not associated with telomere shortening, is called premature senescence. Aging is associated with various age-related complications such as diabetes, osteoporosis, cardiovascular disease, dementia, neurodegenerative disease, renal failure and sarcopenia. Also interesting, aging is the single greatest risk factor for cancer (McHugh et al, J.cell biol.217 (1): 65-77, 2018; childs et al, nat. Rev. Drug discovery.16 (10): 718-735, 2017).

Senescent cells retain metabolic activity and can affect tissue hemostasis, disease and senescence through their secretory phenotype (He et al, cell 169 (6): 1000-1011, 2017). Aging is considered a physiological process that is important in promoting wound healing, tissue homeostasis (Brighton et al, elife 6, 2017), regeneration, embryogenesis, regulation of fibrosis, etc. (von Kobbe, cell mol. Life sci.2018). For example, transient induction of senescent cells is observed during wound healing, and it contributes to wound healing. Perhaps one of the most important effects of aging is its role in tumor genesis inhibition (von Kobbe, cell mol. Life sci.2018). However, aging cell accumulation also drives aging and aging-related diseases. The senescent phenotype may also trigger a chronic inflammatory response and thus enhance a chronic inflammatory condition to promote tumor growth. The link between aging and aging was initially based on the observation that aging cells accumulated in aging tissues. Over the last decade, our understanding of the deleterious consequences of aging in aging and age-related pathologies has been greatly expanded. The use of transgenic models enables systematic detection of senescent cells in many age-related pathologies. The development of genetic and senescent lytic drug strategies that selectively eliminate senescent cells has shown that senescent cells can indeed play a causal role in aging and related pathologies.

Senescent cells exhibit important and unique characteristics including changes in morphology, chromatin organization, gene expression, and metabolism. There are several biochemical and functional properties associated with cellular aging, such as (i) inhibitors of cyclin-dependent kinases, p16 ^INK4a And p21 ^CIP1 (ii) the presence of a marker of aging-related β -galactosidase, i.e., lysosomal activity, (iii) the occurrence of aging-related down-regulation of heterochromatin foci and lamin B1 levels, (iv) resistance to apoptosis caused by increased expression of anti-apoptotic BCL family proteins, and (v)CD26 (DPP 4) (Kim et al, genes Dev.31 (15): 1529-1534, 2017), CD36 (Scavenger) receptor) (Chong et al, EMBO Rep.19 (6), 2018), fork-box 4 (FOXO 4) (Bourgeois et al, FEBS Lett.592 (12): 2083-2097, 2018) and secretory carrier membrane protein 4 (SCAMP 4) (Kim et al, genes Dev.32 (13-14): 909-914, 2018) up-regulated, (vi) lipofuscin accumulation, and (vii) expression of embryonic chondrocyte expression 1 and decoy death receptor 2. Senescent cells also express an inflammatory feature, the so-called SASP. By SASP, senescent cells produce a wide range of inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8, TNF- α), growth factors (TGF-. Beta.0, PDGF-AA, insulin-like growth factor binding proteins (IGFBPs)), chemokines (CCL-2, CCL-20, CCL-7, CXCL-4, CXCL1 and CXCL-12), and matrix metalloproteinases (MMP-3, MMP-9), which operate in a cellular autonomous manner to enhance senescence (autocrine effects), and communicate with and alter the microenvironment (paracrine effects) (Milanovic et al, nature (7686): 96-100, 2018). IL-1α is considered one of the major modulators of SASP. The release of IL-1 alpha by senescent cells transmits senescence to normal cells. IFN can also induce senescence by triggering DNA damage in target cells. IGFBs modulate the insulin-like growth factor (IGF) pathway and IGF acts as a potent senescence inducer. TGF-. Beta.1 secreted as one of the SASP factors can induce and maintain the senescent phenotype and age-related pathological conditions in an autocrine/paracrine fashion. Integrin beta 3 regulated by the polycom protein CBX7 is up-regulated during senescence, promotes senescence by activating TGF-beta signaling in an autocrine/paracrine manner, and enhances SASP in human fibroblasts. Furthermore, TGF- β mediated aging cell accumulation is thought to be associated with idiopathic pulmonary fibrosis. A recent report suggests that TGF- β signaling induces a decrease in H4K20me3 abundance by up-regulating miR-29a/c and down-regulating its target in Suv4-20H in fibroblasts, thereby compromising DNA damage repair and restoration and promoting aging. This pathway leads to in vivo cardiac aging, inhibiting TGF- β signaling can restore H4K20me3 and improve cardiac function in older mice.

Matrix Metalloproteinases (MMPs) are important components of SASP, including MMPs-1 and-3, which can act as regulatory elements of aging. They can cleave IL-8, IL-1, VEGF and other CXCL/CCL family chemokines. In addition, senescent cells secrete serine proteases, such as urokinase or tissue-type plasminogen activators.

SASPs are also composed of non-macromolecular elements such as nitric oxide and reactive oxygen species, which can affect the phenotype of neighboring cells.

The secretory profile of senescent cells depends on the circumstances. For example, mitochondrial dysfunction-related aging (MiDAS) induced by different mitochondrial dysfunctions in human fibroblasts causes the appearance of SASP lacking IL-1 dependent inflammatory factors (Wiley et al, cell Metab.23 (2): 303-314, 2016). The decrease in the NAD+/NADH ratio activates AMPK signaling, which induces MiDAS by activating p 53. As a result, p53 inhibits NF- κb signaling, a key inducer of pro-inflammatory SASP (Salminen et al, cell signal.24 (4): 835-845, 2012). In contrast, cellular senescence caused by sustained DNA damage in human cells induces inflammatory SASP, which relies on the activation of Ataxia Telangiectasia Mutated (ATM) kinase, and not on the activation of p53 (Rodier et al, nat.cell biol.11 (8): 973-979, 2009). In particular, the expression and secretion levels of IL-6 and IL-8 are increased. Cellular senescence caused by ectopic expression of p16.sup.INK4a and p21.sup.CIP1 was also revealed to induce the senescence phenotype of human fibroblasts, and the absence of inflammatory SASP, indicating that growth arrest itself does not stimulate SASP (Coppe et al, J.biol. Chem.286 (42): 36396-36403, 2011). These suggest that the senescence phenotype plays a crucial role in controlling the nature of SASP and its physiological and pathological consequences.

Thus, multiple components of SASP can drive senescence in paracrine fashion in nearby non-senescent cells, thereby increasing the total number of senescent cells. By SASP, senescent cells can also affect the tissue microenvironment through paracrine mechanisms, thereby affecting the recruitment and activation of immune cells in neighboring proliferating cells as well as in aging tissues and tumors.

SASP factors may promote tumor suppression by triggering senescence monitoring, i.e., immune-mediated senescent cell clearance. However, chronic inflammation is also a known driver of tumorigenesis, and there is increasing evidence that chronic SASP can also promote cancer and aging-related diseases. Recently, it has also been shown that senescent cells affect neighboring cells by direct intercellular protein transfer (Biran et al, genes Dev.29 (8): 791-802, 2015). Proteins transferred from senescent cells to adjacent cells of the recipient trigger activation of signal transduction pathways in these cells, resulting in a change in their cellular behavior. A recent study has shown that chemotherapy-induced senescent cancer cells phagocytose neighboring senescent or non-senescent cancer cells. Phagocytosis occurs even in the presence of the cell death inhibitor p 53. The ingested cells degrade in lysosomes. Senescent cells that eat neighbors survive longer in vitro than those that do not. This suggests that metabolic building blocks recovered from lysosomal digestion of neighboring cells are used by senescent cells to increase their survival. Phagocytosis is primarily by phagocytosis rather than by the mechanism of apoptosis (entosis). It has been suggested that cell self-feeding may affect cancer progression by supporting the SASP response. However, this newly acquired ability of chemotherapy-induced aging cancer cells can directly promote or promote cancer cell metastasis by removing specific cells from the tumor microenvironment. If normal cells are found to be also removed by senescent cells in senescent tissue, this may directly lead to tissue degradation.

In summary, all components of SASP are beneficial to the local inflammatory environment and may lead to inflammatory phenomena.

Most of the SASP components are regulated by the nuclear factor kappa light chain enhancer of activated B cells (NF-. Kappa.B), CCAAT/enhancer binding protein beta (CEBP/beta), and mTOR. The transcription factor GATA4 acting upstream of NF-. Kappa.B is also necessary for senescence establishment and SASP induction. Another regulator of SASP is bromodomain and terminal ectodomain (BET) family member bromodomain-containing protein 4 (BRD 4), which positively regulates the senescence secretory protein group and promotes senescence immune clearance. SASPs are also regulated by signal transduction and transcription activator 3 (STAT 3) in certain tissues. Furthermore, mixed lineage leukemia 1 (MLL 1) has been reported to be SASP-enabled, primarily by inducing genes required for DNA replication and DDR activation. Other SASP modulators include NOTCH1 and high mobility group B proteins (HMGB 1 and HMGB 2). Recent data also indicate that SASP can be controlled by the cGAS/STING pathway. cGAS is a DNA sensor that triggers cellular senescence and controls gene transcription of SASP by the adapter protein STING.

One of the most important features of aging is stable growth arrest. This is by p53/p21 ^CIP1 p21 ^cip1 And p16 ^INK4a Rb pathway (McHugh et al, J.cell biol.217 (1): 65-77, 2018). DNA damage and/or DNA Damage Response (DDR) tightly control both pathways.

(1)p53/p21 ^CIP1 p21 ^cip1 : p53 plays a key role in cell senescence, and its activation can be DDR dependent or DDR independent. In the case of telomere DDR dependence, excessive activation of oncogenes and inactivation of oncogenes (oncogene-induced senescence, OIS) caused by telomere depletion, DNA damage and replicative stress activate the DNA damage repair cascade. DDR activates ataxia-telangiectasia mutated kinase (ATM) or ataxia telangiectasia and Rad 3-related (ATR) kinase of the pressure sensor. In turn, ATM/ATR activates p53/p21 by phosphorylating p53 and its ubiquitin ligase Mdm2 ^CIP1 The p21cip1 axis, thereby stabilizing p53 levels. P53 is directly phosphorylated in Ser-15 and indirectly phosphorylated in Ser-20 by Chk 1/2. Many recent studies have also shown that several OIS pathways may actually bypass DDR-activated p53p35. These again demonstrate the key role of p53 and p 53-triggered senescence in inhibiting tumorigenesis after the onset of the first mutation.

Stable up-regulation of p21 by p53 protein ^CIP1 .p21 ^CIP1 p21 ^cip1 .p21 ^cip1 (members of the mammalian cyclin-dependent kinase (CDK) inhibitor family) are required for p 53-induced cell cycle arrest at the G1/S or G2/M checkpoints. P21 encoded by CDKN1A gene located on human chromosome 6 ^CIP1 p21cip1 is a potent cyclin-dependent kinase inhibitor (CKI). It binds to and inhibits the activity of cyclin-CDK 2, -CDK1 and-CDK 4/6 complexes and is therefore useful as a modulator of G1-phase and S-phase cell cycle progression. p21 ^CIP1 p21 ^cip1 Genes that also mediate many p53 targets (such as CDC25C, CDC B)Expression is regulated and survival is recruited primarily by the E4F4 complex. p21 ^CIP1 p21 ^cip1 Also promote aging by inhibiting apoptosis. It binds to many apoptotic agents, including many caspases. P21 in senescent cells ^CIP1 The P21cip1 knockout triggers programmed cell death through a caspase-activated cascade. p21 ^CIP1 p21cip1 is also able to induce senescence independently of p53 activity. The results indicate that Chk2 is able to induce the expression of p21cip1 in p 53-deficient cell lines, which contributes to Chk 2-mediated senescence.

(2)p16 ^INK4a /Rb: three tumor suppressor genes are located at the INK4/ARF locus: p16 ^INK4a And ARF, both of which are encoded by CNDN2A gene, and p15 ^INK4b Encoded by the CDKN2B gene. p15 ^INK4b And p16 ^INK4a Is CDKI and p21 ^CIP1 As such, they affect the cell cycle by binding and inhibiting CDK4 and CDK 6. In contrast, ARF inhibits MDM2, allowing for binding to p53/p21 ^CIP1 The path responds interactively. The INK4/ARF locus served as a sensor of senescence. In young normal cells, the INK4/ARF locus is epigenetically silenced by deposition of an inhibitory H3K27me3 marker. H3K27 methylation is controlled by the polycom inhibitory complexes (PRC 2 and PRC 3). Disruption of PRC1 or PRC2 activity by depletion of expression of certain components thereof inhibits p16 ^INK4a And induce aging. During aging, the H3K27 histone demethylase JMJD3 plays a role in removing the repressor markers surrounding the INK4/ARF locus, promoting its induction. INK4/ARF induction can be observed in tissues during natural aging. In particular, p16 ^INK4a Is considered as a biomarker for aging.

In summary, p53 induces cyclin-dependent kinase inhibitor p21 ^CIP1 And blocks the genes of cell cycle progression (CDK 4 and CDK 6) together with p16.sup.INk4a and p15.sup.INk4b. This eventually causes insufficient phosphorylation of retinoblastoma protein (Rb) and arrest of the cell cycle in the G1 phase (McHugh et al, J.cell biol.217 (1): 65-77, 2018).

Although p53/p21 ^CIP1 The pathway appears to play a key role in the initiation of senescence but involves p16 ^INK4a And pathways of the RB family appear to play a central role in the maintenance of aging. A decrease in p53 levels was observed after induction of senescence, whereas p16 ^INK4a This is indicated by the level remaining at a steady high level. It was also shown that down-regulation of p53 in senescent cells has a different effect depending on p16 activity. p53 successfully induces replication and cell growth in cells with low levels of p16.sup.INK4a, whereas in p16 ^INK4a This is not the case in cells with high activity. These findings indicate that p16 ^INK4a Activation of the/Rb pathway is responsible for demarcating between two different stages of aging: early and reversible phases are dominated by p53 activity, and irreversible phases are dominated by p16 ^INK4a the/Rb pathway is induced.

Recently, the cGAS-cGAMP-STING pathway has become an important link between DNA damage and inflammation, cellular senescence and cancer (Tuo et al, j.exp. Med.215 (5): 1287-1299, 2018). This pathway detects cytoplasmic DNA following DNA damage and activates type I IFN and other cytokines. Although both DNase2 and TREX1 rapidly remove cytoplasmic DNA fragments from the nucleus of pre-senescent cells, expression of these dnases is down-regulated in senescent cells, resulting in accumulation of nuclear DNA in the cytoplasm. This resulted in abnormal activation of the cGAS-STING cytoplasmic DNA sensor, which stimulated SASP by induction of IFN- β (Takahashi et al, nature Comm.9:1249, 2018).

Transforming growth factor-beta (TGF- β) is an evolutionarily conserved superfamily of cytokines that mediate a variety of signaling functions to provide tissue-specific control of cell differentiation and proliferation. They also promote or prevent cell death, promote extracellular matrix protein expression, cell motility and invasion, and control cell metabolism.

The human TGF-beta family includes thirty-three genes encoding homodimer or heterodimer secreted cytokines. Members of this family include activin, bone morphogenic proteins, growth differentiation factors, mullen tube inhibitors, nodal and TGF-. Beta.s. TGF-beta family proteins are synthesized as precursor molecules consisting of a signal peptide, a prodomain (for TGF-beta, referred to as latency-related peptide (LAP)) and a mature polypeptide. The removal of the short N-terminal signal peptide allows for protein folding, glycosylation, and processing in subsequent biosynthetic steps during transport from the endoplasmic reticulum to the golgi apparatus. After dimerization via disulfide bonds, furin family proteases proteolytically cleave polypeptides, resulting in the formation of N-terminal long dimer and disulfide-linked LAP and C-terminal short dimer disulfide-linked mature TGF- β. Structural analysis of the latent form of TGF-beta, where LAP and mature TGF-beta remain bound to each other and form a ligand called the Small Latent Complex (SLC), suggests that LAP directly covers the critical amino acids of the C-terminal dimer and is subsequently used to interact with signaling receptors, thereby inactivating the mature TGF-beta dimer. With processing of TGF-beta polypeptides, N-terminal LAP cross-links with other secreted proteins, latent TGF-beta binding proteins (LTBP) via disulfide bonds to form a Large Latent Complex (LLC). LTBP is an extracellular protein that, upon secretion, mediates LLC deposition into the Extracellular Matrix (EMC) through its ability to crosslink with other proteins of the ECM, such as fibronectin and fibrillin. Thus, LTBP provides a scaffold unit that binds latent TGF- β to the ECM. The three latent complexes of TGF- β require an activation mechanism to release the mature ligand; however, only the activation of the TGF- β1 complex is well characterized. Various modes of latent extracellular latent TGF- β activation in physiologically relevant environments, including proteolysis, low pH, reactive oxygen species, binding to other proteins, and mechanical deformation by shear or integrin-mediated cell traction, indicate a tissue-selective mechanism of cell type selectivity that may depend on the signaling environment. In each case, one side of the LAP in SLC is covalently cross-linked to the 8-Cys domain of LTBP through a Cys33 residue, which in turn is linked to the ECM. With this resistance, the conformation of the latent TGF-beta complex can be altered by pulling on the other end of the LAP through integrins, particularly αvβ1, αvβ6 and αvβ8, resulting in the release of active TGF- β1 from LLC. Various proteases are also capable of activating latent TGF-beta. Many studies strongly suggest that physiological activation of latent TGF- β1 requires combined actions of integrins and proteases. It has also been found that the latent TGF-beta complex is not associated with LTBP, but is disulfide-linked to a membrane-associated protein named GARP (also known as LRRC32 or closely related LRRC 33). GARP is expressed primarily in immune cells such as regulatory T cells (tregs). The function of GARP has been extensively studied on tregs, which complex with αvβ8 integrin to release active TGF- β from the cell surface. GARP is shown to be involved in enhancing Treg-mediated peripheral tolerance. In platelets, thrombin cleavage of GARP has also been shown to result in release of active TGF- β1 from the GARP-LAP-TGF- β1 complex.

Once activated from their LAP, all three TGF- β isoforms function through the same heteromeric transmembrane TGF- β receptor complex formed by dimeric TGF- β type I Receptor (RI) alk5 (also known as tβr1) and dimeric TGF- β type II Receptor (RII) tgfβrii. TGF-beta first associates with the homodimer TGF-beta RII. This interaction causes conformational adaptation between the ligand and tgfbetarii by forming a new high affinity binding site for tgfbetari at the interface of the ligand and tgfbetarii. After recruitment of two units of tgfβri, type II receptor kinases phosphorylate serine and threonine residues in the tgfβri membrane proximal subdomain, which are characterized by a short glycine and serine rich motif (GS), and then induce conformational changes that release the immunophilin FKBP12 from the GS domain. This dissociation reduces the inhibitory interactions of the kinase domain with the GS domain and activates the kinase in the type I receptor. After ligand-induced receptor activation, tgfβri then activates effector SMAD through phosphorylation of its two C-terminal serine residues. In particular, type I receptors phosphorylate two different SMAD proteins, SMAD2 and SMAD3 in the case of TGF- β (and other family members such as activin and nodal), or three different SMAD proteins, SMAD1, SMAD5 and SMAD8 in the case of BMP (and some other GDF and other ligand members). These "receptor-activated SMADs" (R-SMADs) then dissociate from the receptor and bind to SMAD4 to form complexes that translocate into the nucleus where they cooperate with high affinity DNA binding transcription factors and co-regulatory factors to activate or repress target genes. SMAD complexes not only direct gene transcription, which then leads to secondary gene expression changes, but also control mRNA splicing, miRNA expression and processing, and epigenetic changes. With further diversity in SMAD complex formation, SMAD signaling pathways are highly versatile, environmentally dependent and nuances in controlling gene expression.

In addition to typical SMAD signaling, TGF- β can also modulate downstream cellular responses in an environmentally dependent manner through other signaling. These include the ERK MAP kinase pathway, JNK and p38 MAP kinase pathway (via TAK 1), PI3-AKT pathway, JAK-STAT pathway, rho- (like) gtpase pathway and intracellular TGF- βi receptor domain signaling pathway. It is now well known that the wide variety and dependence of SMAD functions depends on these atypical pathways.

Since TGF- β controls differentiation of most, if not all, cell lineages and regulates many aspects of cell and tissue homeostasis, deregulation of TGF- β signaling can lead to dysplasia and disease. Accumulated evidence suggests that impairment of TGF- β signaling and modulation of TGF- β ligands in certain cell types leads to cellular aging, cellular degeneration, tissue fibrosis, inflammation, reduced regenerative capacity, and metabolic dysfunction.

TGF-. Beta.1 secreted as one of the SASP factors can induce or accelerate and maintain the senescent phenotype of various cell types, including fibroblasts, bronchial epithelial cells and cancers, in an autocrine/paracrine fashion. Integrin beta 3 regulated by the polycom protein CBX7 is up-regulated during senescence, promotes senescence by activating TGF-beta signaling in an autocrine/paracrine manner, and enhances SASP in human fibroblasts. Furthermore, TGF- β1 mediated aging cell accumulation is shown in idiopathic pulmonary fibrosis. A recent report shows that TGF- β1 signaling induces a decrease in H4K20me3 abundance by up-regulating miR-29a/c and down-regulating the target Suv4-20H in fibroblasts, thereby compromising DNA damage repair and restoration and promoting aging. This pathway leads to in vivo cardiac aging, inhibiting TGF- β signaling can restore H4K20me3 and improve cardiac function in older mice.

TGF-. Beta.1 is used as a senescence driving factor to induce Vascular Smooth Muscle Cell (VSMC) senescence through Reactive Oxygen Species (ROS) -stimulated NF-. Kappa.B signaling pathway activation and expression of SASP factors, including the plasminogen activator inhibitor type 1 (PAI-1, SERPINE 1). PAI-1 is not only a biomarker of cellular senescence but also a necessary and sufficient condition for p53 downstream replication senescence, a key inducer of the senescence process. There is evidence that there is a PAI-1/TGF- β1 positive feed forward mechanism, providing a model by which elevated tissue levels of TGF- β1 stimulate PAI-1 expression during the appearance of the senescent phenotype, which in turn enhances sustained TGF- β1 synthesis, promotes maintenance of the senescent VSMC population, and possibly promotes expansion thereof (Seo et al, am. J. Nephrol.30:481-490, 2009).

It is well known that aging has tumor-inhibiting effects, which delay clinical progression after chemotherapy. The last decade witnessed that understanding of aging biology has advanced a step forward, especially from its tumor-inhibiting properties to complex, dynamic and interactive properties, which can lead to a pro-cancerous effect (pro-oncogenic effects) on neighboring cancer cells, stroma and vasculature in the tumor microenvironment (Hoare et al, ann.rev.cancer biol.2:175-194, 2018). A very excellent study by Milanovic showed that senescent cells also up-regulated important stem cell-associated transcripts in samples from primary B cell chronic leukemia patients (Milanovic et al, nature 553 (7686): 96-100, 2018). Senescent cells have been shown in patients with Acute Myelogenous Leukemia (AML), in which AML blast cells induce a senescent phenotype in stromal cells which in turn promote AML blast cell survival and proliferation by SASP feedback (Abdul-Aziz et al, blood 133 (5): 446-456, 2019). Tumors are thought to grasp pathophysiological programs aimed at growth regulation involved in organ development or tissue repair and "hijack" this process to achieve oncogene performance, rather than creating a new mechanism for tumor progression (Milanovic et al, trends Cell biol.28 (12): 1049-1061, 2018). Epigenetic mechanisms have been described as responsible for senescence induction (H3K 9 demethylase) and subsequent stem Cell (H3K 9 demethylase inhibition) acquisition (Yu et al, cancer Cell 33 (2): 322-336, 2018).

TGF-. Beta.1 triggers epithelial-mesenchymal transition (EMT) by inducing expression of specific transcription factors Snail and Zeb 1/2. EMT provides migration and invasion behavior to cells due to cell adhesion modifications. This process involves the loss of epithelial characteristics and the acquisition of characteristics that lead to movement and invasive properties. EMT represents an important process leading to cancer cell progression and metastasis.

TGF- β1, as an immunosuppressive cytokine, inhibits the function and development of the innate and adaptive immune systems, including macrophages, natural killer cells, dendritic cells, and T cells. Recent in vivo studies have shown that exposure to tissue or tumor derived TGF- β1 can drive the conversion of circulating NK cells to congenital lymphoid cell I (ILC-I) like phenotype characterized by reduced cytotoxic capacity and the acquisition of several ILC1 related surface markers. Interestingly, TGF-. Beta.1 also synergistically acted with IL-15 via the MAPK pathway to drive the conversion of human NK cells to the ILC-1 like phenotype. TGF- β also inhibits human NK cell metabolism through its classical signaling pathway, thereby inhibiting NK cell cytotoxicity. TGF-. Beta.1 also stimulates regulatory T cells that inhibit other lymphocyte functions. These inhibitory functions confer one of many cancer markers that TGF- β1 avoids immune destruction.

Pancreatic Ductal Adenocarcinoma (PDAC) is the most common malignancy of the pancreas, with a very poor prognosis, a five-year survival of 7% and a median survival of less than 11 months. PDACs are highly refractory to all available antitumor pharmacological options. This is a result of a strong fibroproliferative response associated with the progression of PDAC, exhibiting strong activation of pancreatic stellate cells and formation of dense extracellular matrix, leading to hypoperfusion of the tumor and an impenetrable barrier for intravenous infusion of anticancer drugs or chemotherapeutic agents. TGF- β1 promotes PDAC connective tissue proliferation by enhancing the transformation of fibroblasts or endothelial cells into myofibroblasts (also known as cancer-associated fibroblasts (CAF)). Invasive activity is further amplified by infiltrating immune cells and fibroblasts in the tumor microenvironment, which can produce high levels of TGF-beta. TGF-beta induces pro-angiogenic factors, such as vascular endothelial growth factor, that allow PDAC to progress, invade and metastasize. Acyl-coa synthetase long chain 3 (ACSL 3) was found to be up-regulated in PDAC and associated with increased fibrosis. Reduction of PAI-1 secretion by tumor cells by Acsl3 knockout significantly reduced tumor fibrosis and tumor infiltrating immunosuppressive cells, increasing cytotoxic T cell infiltration in mice. The study also found that PAI-1 expression in PDAC correlated positively with markers of fibrosis and immunosuppression and predicted poor patient survival. Since PAI-1 is a key component of SASP and a mediator of cellular senescence and is regulated by TGF- β1, it is conceivable that TGF- β1 would play a role in the ACSL3-PAI-1 signaling axis of tumor-stroma interaction that mediates pancreatic cancer progression.

In fibrotic diseases, excessive deposition of Extracellular Matrix (EMC) proteins can impair tissue integrity and interfere with normal organ function. Fibrosis can occur in any tissue that is subject to chronic injury, but is most commonly found in the kidney, liver, lung and heart. Fibrosis is driven primarily by inflammatory cytokines, including interleukins and TGF- β superfamily members. Many of these ligands are expressed by infiltrating inflammatory cells that are attracted to the damaged tissue. Overexpression of TGF- β1 induces fibrosis by activating canonical (SMAD-based) and atypical (non-SMAD-based) signaling pathways, resulting in activation of myofibroblasts, overproduction of ECM, and inhibition of ECM degradation. Activation of SMAD 2/3 regulates the expression of several pro-fibrotic genes, including collagen (COL 1A1, COL3A1, COL5A2, COL6A1, COL6A3, and COL7 A1), PAI-1, various proteoglycans, integrins, connective tissue growth factors, and matrix metalloproteinases. This can lead to excessive deposition of ECM, thereby damaging local tissue structures.

Although signaling through SMAD pathways is thought to play a central role in TGF- β fibrosis, emerging evidence suggests that Reactive Oxygen Species (ROS) are also involved in regulating TGF- β signaling through different pathways, including SMAD pathways. TGF- β1 increases mitochondrial ROS production in different cell types, thereby mediating TGF- β -induced apoptosis, aging, EMT, fibrotic gene expression, and myofibroblast differentiation. TGF-beta has been shown to induce the expression of several NADPH oxidase enzymes (Nox), including Nox1, nox2 and Nox4 in different cell types, which are a group of heme-containing transmembrane proteins, important for ROS production by both phagocytes and non-phagocytes. Nox 4-derived ROS mediate the fibrosis of TGF- β, which includes fibroblast activation/myofibroblast differentiation, epithelial and endothelial apoptosis, EMT, and expression of fibroblast/pro-fibrotic genes. Increased Nox4 expression was also detected in fibrotic diseases including IPF, which correlates with increased expression of the myofibroblast marker α -SMA, also supporting the role of Nox4 in fibrotic diseases. Various pathways have been shown to be involved in the induction of Nox4 by TGF- β. These include the SMAD pathway, PI3K pathway, MAPK pathway, and RHOA/ROCK pathway.

Emerging evidence suggests an interactive response between mitochondria and NADPH oxidase. Mitochondrial-derived ROS help increase NOX expression in response to TGF- β, whereas NOX-produced ROS can lead to mitochondrial dysfunction and increase mitochondrial ROS production. The interaction between mitochondria and Nox enzymes has also been shown to mediate the pro-fibrotic effects of TGF- β. Involves a feed-forward interaction between mitochondria and Nox4 in TGF-beta induced ROS production (Jain et al, journal of Biological chemistry 288:770-777, 2013).

By 2030, more than 20% of the population will reach 65 years old or older (see Census/content/dam/Census/library/publications/2014/demo/p 23-212. Pdf), about 40% of the population will be obese (Finkelstein et al, am.j.prev.med.42 (6): 563-570, 2012). Metabolic diseases can affect the ability of a cell to carry out important processes involving protein, carbohydrate and lipid transport or processing. Aging and obesity are major risk factors for chronic diseases susceptible to diabetes, cardiovascular disease and hepatic steatosis, all of which are leading causes of death and thus constitute a significant public health problem (Must et al, "The Disease Burden Associated with Overweight and Obesity" In: feingold KR, anawalt b., boyce a., et al, eds., endotext, south dartrim (MA), 2000; martin et al, nat. Rev. Cardiol.14 (3): 132, 2017).

Excessive calorie intake promotes oxidative stress in the adipose tissue of mice and leads to the characteristics of type 2 diabetes, accompanied by expression of senescence markers (e.g., p53, beta-galactosidase) in mice (Minamino et al, nat. Med.15 (9): 1082-1087, 2009). Aging also promotes biological deterioration of adipose tissue by preventing adipogenic differentiation (Mitterberger et al, gerontol. A biol. Sci.69 (1): 13-24, 2014). A recent further study showed that obesity-induced aging caused anxiety and nerve damage by increasing fat deposition in the brain, and that the removal of these aging cells resulted in an improvement in anxiety-like behavior caused by obesity in mice (Ogrodinik et al, cell Metab.29 (5): 1061-1077, 2019). Other studies have shown that obesity also impairs immune cell function. NK cell effector function was shown to be impaired by lipid accumulation in these cells, and reversal of this process restored function (Michelet et al, nat. Immunol.19 (12): 1330-1340, 2018). Additional studies have shown that NK cell damage in obesity is age-independent, as similar defects are observed in young and older obese individuals (Tobin et al, JCI Insight 2 (24): e94939, 2017; michelet et al, nat. Immunol.19 (12): 1330-1340, 2018).

In mice, increased calorie intake results in fat deposition in blood vessels, which in turn recruits monocytes that phagocytose these lipids and transform into foam macrophages, eventually accumulating in the subendothelial space resulting in atherosclerotic plaques (Bennett et al, nat. Rev. Cardiol.14 (3): 132, 2017; katsuumi et al, front. Cardiovic. Med.5:18, 2018). Mice fed western high fat diet (42% of calories in diet from fat) also showed that the burden of senescent cells was proportional to plaque (lipid-loaded macrophages) formation. Successful elimination of these senescent cells in transgenic mice resulted in a significant reduction in plaque formation (Childs et al, science 354 (6311): 472-477, 2016).

Age, obesity and other factors associated with changes in glucose levels, growth hormone (IGF) can lead to Diabetes (Palmer et al, diabetes 64 (7): 2289-2298, 2015). In mice fed high fat diet, upregulation of senescence markers such as p53 is associated with insulin resistance, whereas inhibition of p53 activity in adipose tissue leads to a decrease in senescence markers and is associated with improvement of insulin resistance in the mouse model (Minamino et al, nat. Med.15 (9): 1082-1087, 2009). Meanwhile, pancreatic β cell aging has been shown to be a factor in the development of type 2 diabetes in obese mice (Sone et al, diabetes 48 (1): 58-67, 2005).

Under high fat feed conditions, the hypothalamus produces excess TGF- β. This can lead to hypothalamic inflammation, hyperglycemia and glucose intolerance. The data indicate that excess TGF- β induces hypothalamic RNA stress response, resulting in accelerated decay of the mRNA of ikbα. IκBα is an inhibitor of NF- κB (Yan et al Nature Medicine 20:1001-1008, 2014). Thus, TGF- β signaling exacerbates obesity and diabetes through effects on the peripheral and central nervous systems.

Aging is a major risk factor for the development of many neurodegenerative diseases. Accumulation of senescent cells in the nervous system has been shown to be associated with Aging and neurodegenerative diseases, and may predispose humans to developing neurodegenerative diseases or may exacerbate their course (krithis et al, int.j.mol. Sci.19 (10:2937, 2018.) cellular Aging may hinder cellular function by: promoting chronic inflammation (Huell et al Acta neuropathol.89 (6): 544-551, 1995; nelson et al, aging Cell 11 (2): 345-349, 2012), 2. Neuronal regeneration depletion (ciprini et al, cereb.cortex28 (7): 2458-2478, 2018), 3. Loss of function (De Stefano et al, j. Neurol. Neurosg. Psychiatry 87 (1): 93-99, 2016) and 4. Blood brain barrier dysfunction (Yamazaki et al, stroke 47 (4): 1068-1077, 2016). Studies have shown that amyloid-containing aβ peptide and misfolded tau protein accumulate in the most common neurodegenerative disease in humans (alzheimer's disease) (Musi et al, aging Cell 17 (6): e12840, 2018). These changes ultimately affect neurons, leading to cognitive impairment and neurodegeneration. High expression of the well-known markers CDKi p16 k4A and MMP-1 and IL-6 (b.6): 9; patent No. 9, patent (fig. 9) and IL-6 (fig. 9) are well-known markers CDKi 4A, e, fig. 2012 (fig. 9, fig. 2012) and fig. 9, fig. 35, fig. 9, fig. 5, fig. 9, 2017). Recent studies have shown that the presence of senescent cells is associated with neuronal disease in animal models (Crews et al, hum. Mol. Genet.19 (R1): R12-R20, 2010; chinta et al, cell Rep.22 (4): 930-940, 2018). Animal model studies reflecting human AD show encouraging results. Clearance of senescent cells from transgenic mice prevents neurofibrillary tangles and abnormal accumulation of tau protein within neurons, thereby preserving cognitive function (Bussian et al, nature 562 (7728): 578-582, 2018). Patients with the second most common neurodegenerative disease, parkinson's Disease (PD), exhibit loss of motor control due to loss of neurons that produce dopamine in the substantia nigra. The most abundant cell type in the CNS, astrocytes, are important for providing structural and metabolic support to neurons and also play a role in controlling the blood brain barrier and blood flow. A recent breakthrough study showed the senescent phenotype of astrocytes in brain samples after PD patients had died (Chinta et al, cell Rep.22 (4): 930-940, 2018). The study also developed an animal model of PD induced by environmental neurotoxin (paraquat), which induces senescence by oxidative stress, which model shows neuropathology associated with PD. The authors showed that the elimination of senescent cells in transgenic mice resulted in the abrogation of paraquat-induced neuropathology.

The aging of human skin may be: 1. intrinsic (chronological), which is the result of physiological and genetic changes over time, or 2. Extrinsic; caused by exposure to external factors such as Ultraviolet (UV) radiation, environmental toxins, and other agents that induce DNA damage (Cavinatoo et al, exp. Gerontol.94:78-82, 2017). In affecting changes in skin tissue with age, the loss of elastic properties caused by changes in elastin production, increased degradation and/or processing has a significant impact on tissue aesthetics and health (Wang et al, front. Genet.9:247, 2018). Acute uv exposure can lead to sunburn, abnormal pigmentation, subcutaneous vascular manifestations (telangiectasias) and immunosuppression, whereas long-term exposure can lead to premature skin aging and even risk of malignancy (ritlie et al, cold Spring harbor. Personal 5 (1): a015370, 2015). There is a direct link between the development of medicine and population growth, which is characterized by an increased number of middle aged and elderly people, thus a significant need for anti-aging treatment (Weihermann et al, int. J. Cosmet. Sci.39 (3): 241-247, 2017). UVB from sunlight has mutagenicity and is directly introduced during DNA replication Causing DNA damage. The sign of photodamaged skin is the accumulation of amorphous elastic fibers and disordered dermal collagen. Studies have shown that this may be due to impaired elasticity and production of fibril proteins, or increased degradation of Matrix Metalloproteinases (MMPs) secreted by senescent cells that have undergone DNA damage (Pittayapruek et al, int.j.mol. Sci.17 (6): 868, 2016). Reactive Oxygen Species (ROS) produced upon UVB radiation activate aging core factors such as nuclear factor-kappa (NF- κB) and mitogen-activated protein kinases (MAPKs) (Pittayapruek et al, int. J. Mol. Sci.17 (6): 868, 2016). UVB irradiation alters TGF-beta signaling pathways in human dermal fibroblasts primarily by reducing the synthesis of transforming growth factor-beta receptor II (tβrii) (Purohit et al, j. Dermotol.83 (1): 80-83, 2016). Several studies have shown the presence of senescent cells in both in vitro and in vivo, as well as in skin exposed to ultraviolet light. Keratinocytes and skin fibroblasts have been widely studied as models of photoaging that express markers of aging, such as p16 ^INK4asd Beta-galactosidase, lamin B1 and senescence-associated secretion phenotype (SASP) (Waaijer et al, aging 10 (2): 278-289, 2018; dimri et al, proc. Natl. Acad. Sci. USA 92 (20): 9363-9367, 1995; wang et al, sci. Rep.7 (1): 15678, 2017; ghosh et al, J. Invest. Dermatol.136 (11): 2133-2139, 2016). Since it is known that senescent cells express NK ligands, induction of NK cells and activation of other immune cells (T regulatory cells) will represent an attractive strategy for clearing senescent cells and maintaining healthy skin (Carr et al, clin. Immunol.105 (2): 126-140, 2002; ali et al, immunology 152 (3): 372-381, 2017).

The demonstration that selective killing of senescent cells significantly improves the healthy life of mice under normal aging conditions and improves the outcome of age-related disease or cancer treatment has stimulated interest in identifying compounds that can clear senescent cells. In nature, senescent cells are often removed by innate immune cells. The induction of senescence not only prevents the potential proliferation and transformation of damaged/altered cells, but also promotes tissue repair by generating SASP factors (Munoz-Espin et al, nat. Rev. Mol. Cell biol.15 (7): 482-496, 2014) that act primarily as chemoattractants for Natural Killer (NK) cells (e.g., IL-15 and CCL 2) and for macrophages (e.g., CFS-1 and CCL 2). These innate immune cells mediate immune surveillance mechanisms that eliminate stressed cells. Senescent cells generally up-regulate NK cell activating receptors NKG2D and DNAM1 ligands, which belong to the stress-inducible ligand family: important components of first-line immunity against infectious diseases and malignancies. Upon receptor activation, NK cells can then specifically induce death of senescent cells through their cytolytic machinery. NK cells have been implicated in liver fibrosis (Sagiv et al, oncogene 32 (15): 1971-1977, 2013), hepatocellular carcinoma (Iannello et al, J.exp. Med.210 (10): 2057-2069, 2013), multiple myeloma (Soriani et al, blood 113 (15): 3503-3511, 2009), and glioma cells stressed by mevalonate pathway dysfunction (Ciaglia et al, int. J.cancer 142 (1): 176-190, 2018). In cancer, combination chemotherapy has been shown to up-regulate senescence markers and NK ligands on KRAS mutant lung tumors, suggesting that NK cells are required for targeting these cells (Rusetti et al, science 362 (6421): 1416-1422, 2018). Endometrial cells undergo acute cellular senescence and do not differentiate into decidua cells. Differentiated decidua cells secrete IL-15 and thereby recruit uterine NK cells to target and eliminate undifferentiated senescent cells, thus helping to remodel and rejuvenate the endometrium (bright on, elife 6, 2017). In a similar mechanism, senescent liver satellite cells expressing p53 bias polarization of resident Kupfer (Kupfer) macrophages and newly infiltrated macrophages towards the pro-inflammatory M1 phenotype during liver fibrosis, which shows senescent cell lysis activity. F4/80+ macrophages have been shown to play a critical role in the clearance of mouse uterine senescent cells to maintain postpartum uterine function (Lujambrio et al, cell 153 (2): 449-460, 2013).

Strategies for senescent cell clearance fall into three main categories: senescent Cell lysis (senolytics), immunotherapy and SASP inhibition (He et al, cell 169 (6): 1000-1011, 2017). There is growing body evidence that senescent cell lysis eliminates senescent cellsEfficacy. In general, senescent cell lysis works by targeting the senescent cell anti-apoptotic pathway (SCAP), e.g., the BCL-2 family of proteins, p53/p21 ^CIP1 p21 axis, PI3K/AKT, receptor tyrosine kinase and HSP90 proteins. In mice, senolytics (senolytics) alleviates a range of conditions associated with the effects of senescent cells. To date, these include effects on heart, blood vessels, metabolism, nerves, radiation-induced, chemotherapy-induced, kidney and lung function, and mobility and weakness in several animal models (Kirkland et al, EBiomedicine 21:21-28, 2017). Many additional senescent cell lysis drugs are currently being developed. Recently, a FOXO 4-related peptide was described that inhibits the PI3K/AKT/p53/p21 pathway and showed encouraging results in human fibroblasts and mouse models in vitro. Other senescent cell lysis methods include ABT-737 and ABT-263 (Tse et al, cancer Res.68 (9): 3421-3428, 2008) acting on BCL-2 protein and A1331852 and A1155463 (Zhu et al, aging (Albany NY) (3): 955-963, 2017) targeting BCL-XL pathway, dasatinib (dasatinib) and quercetin (quercitin) targeting tyrosine kinases have demonstrated senescent cell clearance (Farr et al, nat. Med.23 (9): 1072-1079, 2017). BCL-2 family inhibitors may potentially cause side effects such as neutropenia and thrombocytopenia. Since many senolytics are only in the preclinical stage, it is necessary to study possible side effects before entering clinical stage trials.

Blocking the SASP factor is an alternative strategy to prevent the deleterious effects of senescent cells. These factors include inflammatory chemokines and cytokines, growth factors and matrix remodeling proteases. The central pathways involved in these actions are the NF-. Kappa.B and C/EBP. Beta. Pathways. mTOR inhibitors such as rapamycin (rapamycin) and analogs thereof can eliminate SASP by reducing the expression of membrane-bound IL-1 alpha. Two other notable drugs used to inhibit the NF- κB and C/EBP β pathways in the in vivo mouse model are Metformin (Metformin) and Ruxolitinib (Ruxolitinib) (Moiseeva et al, aging Cell 12 (3): 489-498, 2013; xu et al, proc. Natl. Acad. Sci. USA 112 (46): E6301-6310, 2015), respectively. Other drugs, such as siltuximab or tosituzumab, block cytokines, such as IL-6, another SASP factor. Also, as with certain senolytics, treatment with anti-inflammatory drugs may produce potential side effects (Karkera et al, prostate 71 (13): 1455-1465, 2011). A recent phase I clinical trial using senolytics (dasatinib plus quercetin) did not yield any conclusive results in patients with pulmonary fibrosis (just et al, EBiomedicine 40:554-563, 2019).

The third strategy is immune-mediated intervention, possibly superior to the above strategy. As described above, cells recruited to clear senescent cells include NK cells, macrophages and neutrophils. Senescent cells recruit NK cells primarily through up-regulation of ligands for NKG2D (expressed on NK cells), chemokines and other SASP factors. In vivo models of liver fibrosis have shown that activated NK cells effectively clear senescent cells (Krizhanovsky et al, cell 134 (4): 657-667, 2008). Senescent cells resist NK cell mediated clearance by upregulating the decoy receptor DCR2, DCR2 inhibits apoptosis and limits their clearance primarily through granzyme and perforin mediated pathways (Sagiv et al, oncogene 32 (15): 1971-1977, 2013). Recent data indicate that lipid accumulation in NK cells observed in obese individuals leads to reduced frequency and effector cytotoxic function, and this is age independent (Michelet et al, nat. Immunol.19 (12): 1330-1340, 2018; tobin et al, JCI Insight 2 (24): e94939, 2017). NK cell mediated antibody dependent cytotoxicity (ADCC) has been demonstrated against dipeptidyl peptidase 4 (DPP 4/CD 26) in human senescent cells in vitro, a recently described marker of senescence (Kim et al, genes Dev.31 (15): 1529-1534, 2017). Other strategies include the use of CAR-T cells to alter immune responses against senescent cells (Grupp et al, N.Engl. J. Med.368 (16): 1509-1518, 2013; yousefzadeh et al, nature, published online at 2021, 5, 12).

Studies have described various models of aging including liver fibrosis (Krizhanovsky et al, cell 134 (4): 657-667, 2008), osteoarthritis (Xu et al, j. Gelotol. A biol. Med. Sci.72 (6): 780-785, 2017), parkinson's disease (Chinta et al, cell rep.22 (4): 930-940, 2018), obesity-induced anxiety disorders (ognodnik et al, cell meta ab.29 (5): 1061-1077, 2019), atherosclerosis (child et al, science 354 (6311): 472-477, 2016) and diabetes (Sone et al, diabetes 48 (1): 58-67, 2005). A recent study has shown that transplantation of cells induced by in vitro senescence into young mice results in physical dysfunction (Xu et al, nat. Med.24 (8): 1246-1256, 2018). The question is what kind of therapy is effective in eliminating senescent cells in different tissues. Most available data are based on in vitro experiments and a few mouse studies (Krizhanovsky et al, cell 134 (4): 657-667, 2008; xu et al, nat. Med.24 (8): 1246-1256, 2018; baker et al, nature 479 (7372): 232-236, 2011; farr et al, nat. Med.23 (9): 1072-1079, 2017; xu et al, J. Gerontol. A biol. Sci. Med. Sci.72 (6): 780-785, 2017; bourgeis et al, FEBS Lett.592 (12): 2083-2097, 2018). NK cells offer an attractive strategy to combat the accumulation of senescent cells. However, few studies on aging models have explored this strategy (Krizhanovsky et al, cell 134 (4): 657-667, 2008). Various clinical trials have shown that the treatment of various forms of cancer using adoptive transfer of NK cells was successful (Sakamoto et al, J. Transl. Med.13:277, 2015; miller et al, blood 105 (8): 3051-3057, 2005; cifaldi et al, trends mol. Med.23 (12): 1156-1175, 2017; li et al, cytotherapy 20 (1): 134-148, 2018). Of importance is a recent clinical trial using autologous expanded NK cells in colon cancer patients (Li et al, cytotherapy 20 (1): 134-148, 2018). The authors showed that NK cell therapy combined with chemotherapy prevented relapse and prolonged survival and that side effects were acceptable (Li et al, cytotherapy 20 (1): 134-148, 2018). NK cells activated by cytokine transfer cytokines such as IL-15, IL-12, IL-18 and IL-21 can be used as potential immunotherapeutic strategies for the depletion of senescent cells with minimal side effects (Romee et al, blood 120 (24): 4751-4760, 2012; song et al, eur. J. Immunol.48 (4): 670-682, 2018). Furthermore, the safety of use of NK cells has been demonstrated in acute myelogenous leukemia (Romee et al, blood 120 (24): 4751-4760, 2012; fehniger et al, biol. Blood Marrow Transmount.2018). Other approaches are SASP factors that block circulation, such as TGF-beta, IL-8, and IL-6 (Ganesh et al, immunity 48 (4): 626-628, 2018; georgilis et al, cancer Cell 34 (1): 85-102, 2018). The aging model mentioned above would be the ideal choice for testing these methods. Therefore, more consideration should be given to avoiding adverse side effects caused by the use of foreign compounds and drugs as a strategy to address age-related disorders.

Cell senescence is a series of progressive and phenotypically diverse cellular states obtained after initial growth arrest (Van Deurs, nature 509 (7501): 439-446, 2014). Thus, senescent cells are heterogeneous populations of cells that have few common core properties (Dou et al, nature 550 (7676): 402-406, 2017). Thus, it is difficult to determine a co-senescent cell lysis drug (senolytic drug) target. This also hampers the achievement of the goal of developing senolytics, a method of selectively, safely and effectively eliminating senescent cells after systemic administration. As described above, immune cells are effector cells that naturally eliminate senescent cells after they have completed their physiological roles (Brighton et al, elife 6, 2017). Attenuation of the immune system during aging causes the aging cells to accumulate (Karin et al, nat Commun 10 (1): 5495, 2019) (Chambers et al, allergy Clin Immunol 145 (5): 1323-1331, 2020). Furthermore, TGF-. Beta.is a component of senescent SASP, which when overproduced in tissues, plays a corrosive role in cellular senescence and aging-related pathologies (Tominga et al, int.J.mol. Sci.20 (20), 2019). Provided herein are methods of using complexes of common gamma chain cytokines and their related receptors to promote and activate immune cells, and methods of using TGF- βrii to reduce the amount of active forms of TGF- β in aging tissues and tumor microenvironments by subcutaneous administration to restore their ability to reduce aging cells, and to effectively, selectively and safely reduce chronic inflammation in vivo.

In some embodiments of any of the methods described herein, the method results in regeneration of the aged immune cells in the subject (e.g., one or more of increased metabolic activity (e.g., increased oxidative phosphorylation, increased glycolysis, and increased oxygen consumption) of the aged immune cells in the subject, decreased levels of one or more of p16, p21, and SASP factors in the aged immune cells in the subject, and increased cytolytic activity of the aged immune cells in the subject, e.g., as compared to the level of the subject prior to treatment). As used herein, the term "aged immune cell" refers to an immune cell having one or more of the following: for example, a decrease in metabolic activity (e.g., decreased oxidative phosphorylation, decreased glycolysis, and decreased oxygen consumption) as compared to control immune cells obtained from a healthy subject (non-immunocompromised subject) that is less than half the average life span of the subject population; elevated levels of one or more of p16, p21 and SASP factors; and decreased cytolytic activity. Non-limiting examples of aged immune cells include aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils, aged basophils, aged eosinophils, cumic cells, and aged microglia.

In some embodiments of any one of the methods described herein, the method increases the ratio of naive T cells to memory T cells in the subject. In some embodiments of any one of the methods described herein, the method is such that CD4 in the subject ⁺ T cells and CD8 ⁺ The ratio of T cells decreases.

In some embodiments, regeneration of the aged immune cells results in a reduction in the number of diseased cells or infectious agents in the subject. In some embodiments, the aged immune cells include one or more of aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils, aged basophils, aged eosinophils, aged Kupffer cells, and aged microglia. In some embodiments, the diseased cells include cancer cells, virus-infected cells, and cells infected with intracellular bacteria. In some embodiments, infectious agents include viruses, bacteria, fungi, and parasites.

Also provided herein are methods of using complexes of common gamma chain cytokines and their cognate receptors and/or agents that result in reduced activation of TGF-beta receptors to restore viability of the immune system.

Method for improving the texture and/or appearance of skin and/or hair

Also provided herein are methods of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time (e.g., any of the periods of time described herein), comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators (e.g., any of the NK cell activators described herein or known in the art).

Also provided herein are methods of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time (e.g., any of the periods of time described herein), comprising administering to the subject a therapeutically effective amount of activated NK cells (e.g., any of the activated NK cells described herein or known in the art).

Some embodiments of these methods further comprise: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are haploid NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are allogeneic resting NK cells. In some embodiments of these methods, the resting NK cells are artificial NK cells. In some embodiments of any of these methods, the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

In some embodiments of these methods, the liquid medium is a serum-free liquid medium. In some embodiments of any one of the methods described herein, the liquid medium is a chemically-defined liquid medium. Some embodiments of these methods further comprise isolating the activated NK cells (and further administering a therapeutically effective amount of the activated NK cells to a subject, such as any of the subjects described herein). In some embodiments of these methods, the contacting step is performed for a period of time ranging from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of these methods, the methods result in an improvement in the texture and/or appearance of the skin of the subject over a period of time (e.g., any of the periods of time described herein).

In some embodiments of these methods, for example, the method results in a decrease (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 5% to about 99% (or any subrange of this range described herein) in the subject's skin wrinkle formation rate over a period of time (e.g., any of the time periods described herein) as compared to the subject's wrinkle formation rate prior to treatment or the wrinkle formation rate in a similar subject not receiving treatment.

In some embodiments of these methods, the methods result in an improvement in skin coloration of the subject over a period of time (e.g., any of the periods of time described herein).

In some embodiments of these methods, the methods result in an improvement in the skin texture of the subject over a period of time (e.g., any of the periods of time described herein).

In some embodiments of these methods, the methods result in an improvement in the texture and/or appearance of the subject's hair over a period of time (e.g., any of the periods of time described herein).

In some embodiments of these methods, for example, the method results in a decrease (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 5% to about 99% (or any subrange of this range described herein) in the subject's rate of grey hair formation over a period of time (e.g., any of the time periods described herein) as compared to the rate of grey hair formation in the subject prior to treatment or in a similar subject not receiving the treatment.

In some embodiments of these methods, for example, the method results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 5% to about 99% (or any subrange of this range described herein) in the subject's amount of grey hair over a period of time (e.g., any of the time periods described herein) as compared to the subject's grey hair formation rate prior to treatment or the amount of grey hair in a similar subject who did not receive the treatment.

In some embodiments of these methods, for example, the method results in a decrease (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 5% to about 99% (or any subrange of this range described herein) in the subject's hair loss rate over a period of time (e.g., any of the time periods described herein) as compared to the subject's hair loss rate prior to treatment or the hair loss rate in a similar subject not receiving the treatment.

In some embodiments of these methods, the methods result in the subject's hair texture being improved over a period of time (e.g., any of the periods of time described herein).

In some embodiments of these methods, for example, the method results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 5% to about 99% (or any subrange of this range described herein) in the subject's skin over a period of time (e.g., any of the time periods) compared to the number of aged dermis cells of the subject or the aged dermis cells of a similar subject that has not been treated.

In some embodiments of these methods, improvement in the texture and/or appearance of a subject's skin over a period of time (e.g., any of the time periods described herein) can be assessed using any of the methods described herein or known in the art, including examination of skin lesions, skin tone and pigmentation, skin moisture, temperature, elasticity, and the presence, size, and shape of blood vessels.

In some embodiments of these methods, improvement in the texture and/or appearance of a subject's hair over a period of time (e.g., any of the time periods described herein) can be assessed using any of the methods described herein or known in the art.

In some embodiments of these methods, the period of time is, for example, one month to ten years, one month to nine years, one month to eight years, one month to seven years, one month to six years, one month to five years, one month to four years, one month to three years, one month to two years, one month to eighteen months, one month to twelve months, one month to ten months, one month to eight months, one month to six months, one month to four months, one month to two months one month to six weeks, six weeks to ten years, six weeks to nine years, six weeks to eight years, six weeks to seven years, six weeks to six years, six weeks to five years, six weeks to four years, six weeks to three years, six weeks to two years, six weeks to eighteen months, six weeks to twelve months, six weeks to ten months, six weeks to eight months, six weeks to six months, six weeks to four months, six weeks to two months, two months to ten years, two months to nine years two to eight years, two to seven years, two to six years, two to five years, two to four years, two to three years, two to two years, two to eight months, two to twelve months, two to ten months, two to eight months, two to six months, two to four months, four to ten years, four to nine years, four to eight years, four to seven years, four to six years, four to five years, four to four years, four to three years, four to four months to two years, four to eight months, four to twelve months, four to ten months, four to eight months, four to six months, six to ten years, six to nine years, six to eight years, six to seven years, six to six years, six months to five years, six months to four years, six months to three years, six months to two years, six months to eighteen months, six months to twelve months, six months to ten months, six months to eight months, eight months to ten years, eight months to nine years, eight months to eight years, eight months to seven years, eight months to six years, eight months to five years, eight months to four years, eight months to three years, eight months to two years, from eight months to eighteen months, from eight months to twelve months, from eight months to ten months, from ten months to ten years, from ten months to nine years, from ten months to eight years, from ten months to seven years, from ten months to six years, from ten months to five years, from ten months to four years, from ten months to three years, from ten months to two years, from ten months to eighteen months, from ten months to twelve months, from twelve months to ten years, from twelve months to nine years, from twelve months to eight years, from twelve months to seven years, twelve months to six years, twelve months to five years, twelve months to four years, twelve months to three years, twelve months to two years, twelve months to eighteen months, eighteen months to ten years eighteen months to nine years, eighteen months to eight years, eighteen months to seven years, eighteen months to six years, eighteen months to five years, eighteen months to four years, eighteen months to three years, eighteen months to two years, two years to ten years, a combination of two or more of eighteen months to nine years, eighteen months to eight years, eighteen months to seven years, eighteen months to six years, eighteen months to five years eighteen months to four years, eighteen months to three years, eighteen months to two years, two years to ten years, five to nine years, five to eight years, five to seven years, five to six years, six to ten years, six to nine years, six to eight years, six to seven years, seven to ten years, seven to nine years, seven to eight years, eight to ten years, eight to nine years, or nine to ten years of ears.

In some embodiments of these methods, the subject is aged from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old, from about 95 to about 100 years old, from about 100 to about 105 years old, from about 105 to about 110 years old, from about 110 to about 115 years old, or from about 115 to about 120 years old.

Methods for aiding in the treatment of obesity in a subject

Provided herein are methods of aiding in the treatment of obesity in a subject in need thereof over a period of time (e.g., any of the time ranges described herein), comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators (e.g., any of the NK cell activators described herein or known in the art).

Also provided herein are methods of aiding in the treatment of obesity in a subject in need thereof over a period of time (e.g., any of the time ranges described herein), comprising administering to the subject a therapeutically effective amount of activated NK cells (e.g., any of the activated NK cells described herein or known in the art).

Some embodiments of these methods further comprise: obtaining resting NK cells; and contacting resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein said contacting results in activated NK cell production, followed by administration of said NK cells to a subject. In some embodiments of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are haploid NK cells obtained from the subject. In some embodiments of these methods, the resting NK cells are allogeneic resting NK cells. In some embodiments of these methods, the resting NK cells are artificial NK cells. In some embodiments of any of these methods, the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

In some embodiments of these methods, the liquid medium is a serum-free liquid medium. In some embodiments of any one of the methods described herein, the liquid medium is a chemically-defined liquid medium. Some embodiments of these methods further comprise isolating the activated NK cells (and further administering a therapeutically effective amount of the activated NK cells to a subject, such as any of the subjects described herein). In some embodiments of these methods, the contacting step is performed for a period of time ranging from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of these methods, for example, the method results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 5% to about 99% (or any subrange of this range described herein) in mass of the subject over a period of time (e.g., any of the time periods described herein) compared to mass of the subject prior to treatment.

In some embodiments of these methods, for example, the method results in a decrease (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 5% to about 99% (or any subrange of this range described herein)) in the subject's BMI over a period of time (e.g., any of the time periods described herein) as compared to the subject's Body Mass Index (BMI) prior to treatment.

In some embodiments of these methods, for example, the method reduces (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 5% to about 99% (or any subrange of this range described herein)) the rate of progression from pre-diabetes to type 2 diabetes in the subject as compared to the rate of progression from pre-diabetes to type 2 diabetes in the pre-subject or the rate of progression from pre-diabetes to type 2 diabetes in an untreated, similar subject.

In some embodiments of these methods, for example, the method reduces (e.g., by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, or by at least 95%, or by about 5% to about 99% (or any subrange of this range described herein)) the fasting serum glucose level of the subject compared to the fasting serum glucose level of the subject prior to the treatment.

In some embodiments of these methods, for example, the method increases the insulin sensitivity level of the subject (e.g., by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 99%, or by about 10% to about 500% (or any subrange of this range described herein)) compared to the insulin sensitivity of the subject prior to treatment.

In some embodiments of these methods, for example, the method results in a decrease in the severity of atherosclerosis in the subject (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or about 5% to about 99% (or any subrange of this range described herein)) compared to the severity of atherosclerosis in the subject prior to treatment.

In some embodiments of these methods, the treatment of obesity in a subject over a period of time (e.g., any of the periods of time described herein) can be assessed by any of the methods described herein or known in the art, including, for example, measuring body weight and/or body size, body fat, body or local body fat, and Body Mass Index (BMI).

In some embodiments of these methods, the subject's response to treatment can be monitored by measuring fasting serum glucose levels or glucose tolerance according to standard techniques. In some embodiments of these methods, insulin sensitivity may be measured using any of the methods described herein or known in the art, including hyperinsulinemic normoglycemic jaws and intravenous glucose tolerance tests, in vivo steady state model assessment (HOMA), and quantitative insulin sensitivity check index (quacki).

In some embodiments of these methods, the severity of atherosclerosis in a subject can be measured using any of the methods described herein or known in the art, including cardiac catheterization, doppler ultrasound examination, blood pressure comparison, MUGA/radionuclide angiography, thallium/myocardial perfusion scanning, and computed tomography.

In some embodiments of these methods, the period of time is one month to ten years (or any subrange of this range described herein).

In some embodiments of these methods, the subject is in the age range of about 1 to about 5 years old, about 5 to about 10 years old, about 10 to about 15 years old, about 15 to about 20 years old, about 20 to about 25 years old, about 25 to about 30 years old, about 30 to about 35 years old, about 35 to about 40 years old, about 40 to about 45 years old, about 45 to about 50 years old, about 50 to about 55 years old, about 55 to about 60 years old, about 60 to about 65 years old, about 65 to about 70 years old, about 70 to about 75 years old, about 75 to about 80 years old, about 80 to about 85 years old, about 85 to about 90 years old, about 90 to about 95 years old, about 95 to about 100 years old, about 100 to about 105 years old, about 105 to about 110 years old, about 110 to about 115 years old, or about 115 to about 120 years old.

Additional therapeutic agents

Some embodiments of any of the methods described herein can further comprise administering to the subject (e.g., any of the subjects described herein) a therapeutically effective amount of one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered to the subject substantially simultaneously with the NK cell activator or activated NK cells (e.g., as a single formulation or two or more formulations administered to the subject). In some embodiments, one or more additional therapeutic agents may be administered to the subject prior to administration of the NK cell activator or activation of NK cells. In some embodiments, one or more additional therapeutic agents may be administered to the subject after administration of one or more NK cell activators or activated NK cells to the subject.

Non-limiting examples of additional therapeutic agents include: anticancer drugs, activated receptor agonists, immune checkpoint inhibitors, agents for blocking HLA-specific inhibitory receptors, glycogen Synthase Kinase (GSK) 3 inhibitors and antibodies.

Non-limiting examples of anticancer drugs include antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (capecitabine), cytarabine (cytarabine), floxuridine (floxuridine), fludarabine (fludarabine), gemcitabine (gemcitabine), hydroxyurea, methotrexate, 6-thioguanine, cladribine (cladbabine), nelarabine (nelaradine), penstatin (pentastatin) or pemetrexed)), plant alkaloids (e.g., vinblastine (vinblastine), vincristine (vincristine), vindesine (vindesine), camptothecine (camptothecine), 9-methoxycamptothecine, guanamine, cerulodine (coronoline), paclitaxel, wusanguinarine (naucleaoral), diprenylated indole alkaloid (diprenylated indole alkaloid), shancornflower alkaloid (montanine), shizanine (schischikineine), protoberberine (protoberine), berberine (berberine), sanguinarine (sanguinarine), chelerythrine (cheleynine), chelidonine (celandine), ceriponine (celiodine), beta-carboline, demethoxytyline (antoine), tyline (lonine), chelidonine (lonine), cinine (phylline), sinonine (cine), sinonine (sinonine), sinonine (37ginine, high mountain tennis alkaloids (monobtain), rosacea (elliptidine), paclitaxel (paclitaxel), docetaxel (docetaxel), etoposide (etoposide), teniposide (teniposide), irinotecan (irinotecan), topotecan (topotecan) or acridone alkaloids), proteasome inhibitors (e.g., lactacytocin), disulfiram (distnfiram), epigallocatechin-3-gallate, ma Lizuo m (maritimibe) (salinomycin (salinosporamide) A), oprazole (oprazole) (ONX-0912), dipyridamole (delazomib) (CEP-18770), epothilone (epothilin), MG132, beta-hydroxy beta-methylbutyrate, bortezomib (botzomib), carfil (carfilzomib) or carfil), spinosyn (62), dactinomycin (spinosyn), dactinomycin (e.g., dactinomycin (62), dactinomycin (dactinomycin) or other inhibitors (aureomycin), dactinomycin (dactinomycin), dactinomycin (dactinomycin) or other inhibitors (dactinomycin), dactinomycin (dactinomycin) or dactinomycin (62), dactinomycin (dactinomycin) and other drugs (dactinomycin) and the like Entenostat (entinostat), phenylbutyrate, valproic acid, trichostatin (trichostatin) A, dactylostat (dactinostat), mo Xisi Ta (mocetinostat), prazistat (pracinostat), nicotinamide (nicotonamide), canrenol (cambinol), tenovin (tenovin) 1, tenovin 6, statin (sirtinol), ritodstat (ricolinostat), tefefilostat (tefinostat), kevirin (kevitrarin), quasimestat (quisinostat), lei Minuo Takedinaline (quasimide), cetabamine (chidamide) or rilstat (selistat)), tyrosine kinase inhibitors (e.g., acxitinib (axitinib), dasatinib (dasatinib), enkeafinib (encorafinib), erlotinib (erlotinib), imatinib (imatinib), nilotinib (nilotinib), pazopanib (pazopanib) and sunitinib) and chemotherapeutic agents (e.g., all-trans retinoic acid, azacitidine, azathioprine (azathioprine), doxifluridine, epothilone (epothilone), hydroxyurea, imatinib, teniposide, thioguanine, valrubicin, valrubifenib (vemurafenib) and lenalidomide). Further examples of chemotherapeutic agents include alkylating agents such as, for example, methylene chloride, cyclophosphamide (cyclophosphamide), chlorambucil (chloramamide), melphalan (melphalan), ifosfamide (ifosfamide), thiotepa (thiotepa), hexamethylmelamine (hexamethelamine), busulfan (busulfan), hexamethylmelamine (altamine), procarbazine (procarbazine), dacarbazine (dacarbazine), temozolomide (temozolomide), carmustine (carmustine), robustatin (lumustine), streptozolomycin (streptozolomide), carboplatin (carboplatin), cisplatin (cisplatin) and oxaliplatin (oxaliplatin).

Non-limiting examples of activating receptor agonists include any agonist of activating receptor that activates and enhances the cytotoxicity of NK cells, including anti-CD 16 antibodies (e.g., anti-CD 16/CD30 bispecific monoclonal antibodies (BiMAb)) and Fc-based fusion proteins. Non-limiting examples of checkpoint inhibitors include anti-PD-1 antibodies (e.g., MEDI 0680), anti-PD-L1 antibodies (e.g., BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316, anti-PD-L1/CTLA-4 bispecific antibodies KN046, anti-PD-L1/TGF-beta RII fusion protein M7824, anti-PD-L1/TIM-3 bispecific antibodies LY3415244, atezolizumab, or avermectin), anti-TIM 3 antibodies (e.g., TSR-022, sym023, or MBG 453), and anti-CTLA-4 antibodies (e.g., AGEN1884, MK-1308, or anti-CTLA-4/OX 40 bispecific antibodies ATOR-1015). Non-limiting examples of agents for blocking HLA-specific inhibitory receptors include monalizumab (e.g., anti-HLA-E NKG2A inhibitory receptor monoclonal antibodies). Non-limiting examples of GSK3 inhibitors include tegafur Lu Xibu (tidegrouib) or CHIR99021. Non-limiting examples of antibodies that can be used as additional therapeutic agents include anti-CD 26 antibodies (e.g., YS 110), anti-CD 36 antibodies, and any other antibody or antibody construct that can bind to and activate an Fc receptor (e.g., CD 16) on NK cells. In some embodiments, the additional therapeutic agent may be insulin or metformin (metaformin).

Exemplary methods comprising administering one or more common gamma chain family cytokine receptor activators

Provided herein are methods of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Also provided herein are methods of reducing accumulation of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Also provided herein are methods of reducing the level of a marker of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. In some embodiments, the marker of naturally occurring and/or treatment-induced senescent cells is p21 ^CIP1 p21 and CD26. Additional markers of naturally occurring and/or treatment-induced senescent cells are described herein. Additional markers of naturally occurring and/or treatment-induced senescent cells are known in the art.

Also provided herein are methods of reducing the activity of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

Also provided herein are methods of reducing the level and/or activity of one or more naturally occurring and/or therapeutically induced senescence-associated secretory phenotype (SASP) factors derived from senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. In some embodiments, the senescent cells express an inflammatory feature, wherein the inflammatory feature is a SASP factor. In some embodiments, senescence-associated secretory phenotype (SASP) factors include, but are not limited to, inflammatory cytokines (e.g., IL-1α, IL-1β, IL-6, IL-8, and TNF- α), growth factors (e.g., TGF- β, PDGF-AA, and insulin-like growth factor binding proteins (IGFBPs)), chemokines (e.g., CCL-2, CCL-20, CCL-7, CXCL-4, CXCL1, and CXCL-12), and matrix metalloproteinases (e.g., MMP-3, MMP-9) that operate in a cellular autonomous manner to enhance senescence (autocrine effects) and communicate with and alter microenvironments (paracrine effects). In some embodiments, the method reduces the expression level and/or activity of one or more (e.g., two, three, four, or five) senescence-associated secretory phenotype (SASP) factors. In some embodiments, the level of expression or activity of a SASP factor is determined using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the level of expression or activity of a SASP factor is determined using immunoblotting.

In some embodiments of any of the methods described herein, the subject has been previously diagnosed or identified as having an aging-related disease (e.g., any of the exemplary types of aging-related diseases or conditions described herein or known in the art) or an inflammatory disease (e.g., any of the exemplary types of aging-related diseases or conditions described herein or known in the art).

In some embodiments, the aging-related disorder is inflammatory aging-related.

In some embodiments, the aging-related disease is cancer (e.g., any of the exemplary types of cancers described herein or known in the art).

In some embodiments of any one of the methods described herein, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, crohn's disease, multiple sclerosis, guillain barre syndrome, psoriasis, grave's disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders and cognitive disorders associated with cancer treatment.

In some embodiments of these methods, the treatment-induced senescent cells are chemotherapy-induced senescent cells.

In some embodiments of these methods, for example, administration results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 5% to about 99% (or any subrange of this range described herein)) in the subject's amount of naturally occurring and/or treatment-induced senescent cells in the subject's target tissue (e.g., any of the exemplary types of target tissue described herein) compared to the amount of naturally occurring and/or treatment-induced senescent cells in the subject's target tissue prior to treatment.

In some embodiments of these methods, for example, administration results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 5% to about 99% (or any subrange of this range described herein)) in the subject compared to the naturally occurring and/or therapeutically induced accumulation of senescent cells in the subject prior to treatment or the naturally occurring and/or therapeutically induced senescent cells in a similar subject that did not receive the treatment.

In some embodiments of these methods, for example, administration results in a decrease (e.g., at least a decrease of 5%, at least a decrease of 10%, at least a decrease of 15%, at least a decrease of 20%, at least a decrease of 25%, at least a decrease of 30%, at least a decrease of 35%, at least a decrease of 40%, at least a decrease of 45%, at least a decrease of 50%, at least a decrease of 55%, at least a decrease of 60%, at least a decrease of 65%, at least a decrease of 70%, at least a decrease of 75%, at least a decrease of 80%, at least a decrease of 85%, at least a decrease of 90%, or at least a decrease of 95%, or a decrease of about 5% to about 99% (or any subrange of the ranges described herein)) in the level of one or more of the naturally occurring and/or therapeutically induced markers of senescent cells in the subject as compared to the level of the one or more of the naturally occurring and/or therapeutically induced markers of senescent cells in the subject prior to the treatment.

As used herein, a "naturally occurring senescent cell" is a senescent cell that is produced as a result of normal aging or inflammatory processes. Naturally occurring senescent cells may accumulate in various tissues and organs of an individual over time. Naturally occurring senescent cells can be any of the exemplary types of senescent cells described herein that are not induced by therapeutic treatment (e.g., chemotherapy or radiation therapy).

The "treatment-induced senescent cells" as described herein are senescent cells that are generated as a result of therapeutic treatment (e.g., chemotherapy or radiation therapy).

Cytokine receptor activators of the common gamma chain family

The methods described herein comprise the use or administration of one or more common gamma chain family cytokine receptor activators. In some embodiments, the common gamma chain family cytokine receptor activator is a single chain chimeric polypeptide (e.g., any of the exemplary single chain chimeric polypeptides described herein), a multi-chain chimeric polypeptide (e.g., any of the exemplary multi-chain chimeric polypeptides described herein), a soluble IL-15 or IL-15 agonist (e.g., any of the soluble IL-15 or IL-15 agonists described herein), a soluble IL-2 or IL-2 agonist (e.g., any of the soluble IL-2 or IL-2 agonists described herein), a complex of a common gamma chain family cytokine (or a functional fragment thereof) and an antibody or antigen-binding antibody fragment that specifically binds the common gamma chain family cytokine.

Exemplary Single chain chimeric Polypeptides

A non-limiting example of a common gamma chain family cytokine receptor activator is a single chain chimeric polypeptide comprising: (i) a first target binding domain, (ii) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art), and (iii) a second target binding domain, wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine, an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common gamma chain family cytokine receptor, or an antigen binding domain that specifically binds to a common gamma chain family cytokine.

Some embodiments of any of the single-chain chimeric polypeptides described herein can further comprise one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the N-terminus and/or C-terminus thereof.

In some embodiments of any one of the single chain chimeric polypeptides described herein, one or more of the following is a soluble common gamma chain family cytokine: a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art). Non-limiting examples of soluble common gamma chain family cytokines include soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15, and soluble IL-21.

In some embodiments, one or both of the first target binding domain and the second target binding domain comprises a receptor for a soluble common gamma chain family cytokine (e.g., soluble IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors).

In some embodiments of any one of the single chain chimeric polypeptides described herein, one or more of the following is an agonistic antigen binding domain that specifically binds to a soluble common gamma chain family cytokine receptor: a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art). Non-limiting examples of common gamma chain family cytokine receptors include receptors for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

Multichain chimeric polypeptides

A non-limiting example of a common gamma chain family cytokine receptor activator is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding polypeptide domain; (ii) a soluble tissue factor domain; (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; (ii) A second target binding domain, wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine, an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common gamma chain family cytokine receptor, or an antigen binding domain that specifically binds to a common gamma chain family cytokine.

In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the following are soluble common gamma chain family cytokines: a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art). Non-limiting examples of soluble common gamma chain family cytokines include soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15, and soluble IL-21.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more of the following is an agonistic antigen binding domain that specifically binds to a soluble common gamma chain family cytokine receptor: a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art). Non-limiting examples of common gamma chain family cytokine receptors include receptors for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) of the following are soluble common gamma chain family cytokine receptors: a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art); and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art).

In some embodiments of the multi-chain chimeric polypeptides described herein, the first domain or the second domain of a pair of affinity domains is a soluble common gamma chain family cytokine or an antigen binding domain that specifically binds a common gamma chain family cytokine receptor.

Soluble common gamma chain family cytokines

In some embodiments of any of the single-or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain can be a soluble common gamma chain family cytokine. In some embodiments, the common gamma chain family cytokine receptor activator may be a soluble common gamma chain family cytokine. Non-limiting examples of soluble common gamma chain family cytokines include soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15, and soluble IL-21. Non-limiting examples of sequences for soluble IL-2, soluble IL-7, soluble IL-15, and soluble IL-21 are described herein. Non-limiting examples of soluble IL-4 and IL-9 sequences are shown below.

Human soluble IL-4 (SEQ ID NO: 335)

Human soluble IL-9 (SEQ ID NO: 336)

Antigen binding domains

In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each an antigen binding domain. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is an scFv or single domain antibody (e.g., va _H H or V _NAR Domain).

In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor. In some embodiments, an agonistic antigen binding domain (e.g., any of the antigen binding domains described herein) can specifically bind to a receptor for IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21.

The antigen binding domains present in any of the single-chain chimeric polypeptides or multi-chain chimeric polypeptides described herein are each independently selected from the group consisting of: VHH domain, VNAR domain, and scFv. In some embodiments, any of the antigen binding domains described herein are BiTe, (scFv) ₂ Nanobody, nanobody-HAS, DART, tandAb, single chain diabody (scDiabody), single chain diabody-CH 3, scFv-CH-CL-scFv, HSAbody, single chain diabody-HAS, or tandem scFv. Additional examples of antigen binding domains that can be used in any single or multi-chain chimeric polypeptide are known in the art.

In some embodiments, each antigen binding domain in a single-chain or multi-chain chimeric polypeptide described herein is a VHH domain, or at least one antigen binding domain is a VHH domain. In some embodiments, each antigen binding domain in a single-chain or multi-chain chimeric polypeptide described herein is a VNAR domain, or at least one antigen binding domain is a VNAR domain. In some embodiments, each antigen binding domain in a single-chain or multi-chain chimeric polypeptide described herein is an scFv domain, or at least one antigen binding domain is an scFv domain.

In some embodiments, two or more polypeptides present in a single-chain or multi-chain chimeric polypeptide can be assembled (e.g., non-covalently assembled) to form any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), a VHH-scAb, a VHH-Fab, a dual scFab, a F (ab') 2, a bifunctional antibody, an interchangeable mab, a DAF (two-in-one), a DAF (four-in-one), a Dutamab, a DT-IgG, a mortar-pestle type common light chain, a mortar-pestle type assembly, a charge pair, a Fab arm exchange, a SEEDbody, a LUZ-Y, fcab, a kappa lambda body, an orthogonal Fab, a DVD-IgG, an IgG (H) -sc Fv, scFv- (H) IgG, igG (L) -scFv, scFv- (L) IgG, igG (L, H) -Fv, igG (H) -V, V (H) -IgG, igG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, igG-2scFv, scFv4-Ig, zybody, DVI-IgG, diabody-CH 3, triad body, minibody (minibody), triBi minibody, scFv-CH3 KIH, fab-scFv, F (ab') 2-scFv2, scFv-KIH, fab-scFv-Fc, tetravalent HCAb, single chain diabody-Fc, intrabody, docking and locking (dock and lock), lmmTAC, igG-IgG conjugate, cov-X body, and scFv1-PEG-scFv2. See, e.g., spiess et al, mol. Immunol.67:95-106, 2015, which are incorporated herein in their entirety to describe these elements. Non-limiting examples of antigen binding fragments of antibodies include Fv fragments, fab fragments, F (ab') ₂ Fragments and Fab' fragments. Further examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, igG2, igG3, or IgG 4) (e.g., human or humanized IgG, such as antigen-binding fragments of human or humanized IgG1, igG2, igG3, or IgG 4); an antigen-binding fragment of IgA (e.g., an antigen-binding fragment of IgA1 or IgA 2) (e.g., a human or humanized IgA, such as an antigen-binding fragment of human or humanized IgA1 or IgA 2); antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD); antigen binding fragments of IgE (e.g., antigen binding fragments of human or humanized IgE); or an antigen-binding fragment of IgM (e.g., an antigen-binding fragment of human or humanized IgM).

Soluble IL-15 and IL-15 agonists

Non-limiting examples of common gamma chain family cytokine receptor activators are soluble IL-15 or IL-15 agonists. IL-15 acts through a trimeric IL-15 receptor complex that uniquely binds to the IL-15Rα chain and co-IL-15 Rβ and the γ chain shared with IL-2 (also known as IL-2Rβ/γ) by high affinity that confers receptor specificity to IL-15.

In some embodiments, soluble IL-15 is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO 82. In some embodiments, the soluble IL-15 is recombinant soluble human IL-15. In some embodiments, the soluble IL-15 is a mutant IL-15 having one or more amino acid substitutions as compared to wild-type IL-15 (e.g., SEQ ID NO: 82). For example, mutant IL-15 may include a D8N or D8A amino acid substitution as compared to wild-type IL-15. In some embodiments, soluble IL-15 can be coupled to a polymer (see, e.g., miyazaki et al, processed. Annual Meeting AACR, abstract 3265, 2019).

Some embodiments of IL-15 agonists described herein may include a complex of IL-15 and all or part of a soluble IL-15 receptor (IL-15R). Complexes of IL-15 and fully or partially soluble IL-15R may have an extended half-life and/or higher potency than free IL-15. In some embodiments, an IL-15 agonist described herein further comprises an Fc domain (e.g., any of the exemplary Fc domains described herein).

In some embodiments, the partially soluble IL-15R is IL-15Rα. For example, IL-15 may associate with IL-15Rα -Fc fusion to form an IL-15:IL-15Rα -Fc complex (see Stoklasek et al, J. Immunology 177:6072-80, 2006; dubios et al, J. Immunol.180:2099-106, 2008; eparbaud et al, cancer Res.68:2972-83, 2008; rubenstein et al, proc. Natl. Acad. Sci. U.S.A.103:9166-71, 2006). In some embodiments, soluble IL-15 and IL-15Rα form heterodimers (see, e.g., colon et al, cancer Res.79 (13 journal): CT082, 2019, 7, 1).

In some embodiments, the partially soluble IL-15R is part of an IL-15Rα (e.g., the sushi domain of IL-15Rα).

The IL-15 in the complex may be wild-type IL-15 or mutant IL-15. For example, a mutant IL-15 containing an N72D mutation can be used to complex with all or part of a soluble IL-15R (e.g., the sushi domain of IL-15Rα). In some embodiments, the complex is ALT-803, which includes a human IL-15 mutant IL-15N72D complexed with an IL-15Rα sushi-Fc fusion (see, e.g., zhu et al, J.Immunol.183 (6): 3598-607, 2009).

Non-limiting examples of IL-15 agonists include ALT-803/N-803 (Altor Bioscience)/immunology (ImmunityBio)), BNZ-1 (Bioniz treatment Co (Bioniz Therapeutics)), NIZ985 (Novartis), RTX-212 (ruby treatment Co (Rubius Therapeutics)), AM0015 (rhIL-15) (Gilley), IGM-7354 (IGM), xmAb24306 (Roche/Xencor), KD033 (srKD 033) (Kadmon), OXS-C3550 (GT Biopharma), and NKTR-255 (inner Kketta treatment Co (Nektar Therapeutics)).

Soluble IL-2 and IL-2 agonists

Non-limiting examples of common gamma chain family cytokine receptor activators are soluble IL-2 or IL-2 agonists. IL-2 is a cytokine by which CD4 is bound ⁺ T regulatory cells and cytotoxic effector lymphocytes (e.g., CD 8) ⁺ T cells and NK cells) are mainly involved in immune tolerance and immune activation. IL-2 acts on cells expressing a dimeric IL-2 receptor (IL-2R) consisting of IL-2R beta and gamma chains or a trimeric alpha beta gamma receptor (IL-2R alpha beta gamma), which has a 10-100 fold higher affinity for IL-2 than dimeric IL-2R. CD4 ⁺ T regulatory cells are characterized by strong constitutive expression of IL-2Rα, which enables the cell to express IL-2Rαβγ, thereby using low levels of IL-2. Dimer IL-2R is in CD8 undergoing antigen (memory) ⁺ T cells and NK cells are the most prominent. Thus, in addition to activating Treg cells, high levels of IL-2 strongly stimulate CD8 ⁺ T cells and NK cells.

In some embodiments, the soluble IL-2 is at least 90% (e.g., at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identical to SEQ ID NO: 78. In some embodiments, soluble IL-2 is recombinant human IL-2. The soluble IL-2 may be an IL-2 variant. For example, the binding of an IL-2 variant to IL-2Rβ is more potent (e.g., at least 50, 100, 150, or 200-fold more potent) than to IL-2Rα. An exemplary IL-2 variant is MDNA109 (see, e.g., rafei et al, J.Clin. Oncol.37 (15 journal), 2019). In some embodiments, the IL-2 variant has abrogated CD25 binding. For example, residues F42, Y45 and L72 involved in CD25 binding may be mutated (see, e.g., klein et al, oncominium 6 (3): e1277306, 2017).

In some embodiments, the IL-2 agonist is a pegylated IL-2 that binds to a limited amount of the IL-2Rα subunit and preferentially binds to dimeric IL-2Rβγ (see, e.g., benterbibel et al, cancer discover.9 (6): 711-721, 2019).

Some embodiments of IL-2 agonists described herein are fusion proteins comprising IL-2. In some embodiments, the fusion protein includes IL-2 or a variant thereof linked to all or part of a soluble IL-2R. In some embodiments, the moiety of the soluble IL-2R is IL-2Rα (see, e.g., vaishamayan et al, J.Clin. Oncol.35 (15 journal), 2017). For example, the fusion protein can selectively activate dimeric IL-2Rβγ. Other examples of IL-2 fusion proteins include those fused to a toxin (e.g., diphtheria toxin).

In some embodiments, the fusion protein includes an IL-2 or variant thereof (e.g., any of the IL-2 variants described herein) linked to an antibody (e.g., a monoclonal antibody or scFv). Non-limiting examples of antibodies that can be linked to IL-2 or variants thereof include human monoclonal antibodies against fibroblast activation protein- α (FAP) (see, e.g., soeresen et al, j. Clin. Oncol.36, journal 15), anti-CD 20 monoclonal antibodies (see, e.g., lansigan et al, blood 128 (22): 620, 2016), scFv against the A1 domain of tenascin-C (see, e.g., catania et al, cell adh. Migr.9 (1-2): 14-21, 2015); and anti-CEA antibodies (see, e.g., klein et al, oncoimmunol.6 (3): e1277306, 2017).

Additional examples of IL-2 agonists include the Aldi interleukin (Clinigen), pulmoleukin (immune service), NKTR-214 (Nectron Takara Shuzo Co., ltd. (Nektar Therapeutics)), DI-Leu16-IL2 (Alopex/Source Biopharmaceutical (Provenance Biopharmaceuticals)), RG7461 (Roche), teleukin (Philogen), ALT-801803 (Alter Bioscience), ALT-801 (Alter Bioscience), ALKS 4230 (Alker mes), almer interleukin-2-Tituzumab (cergutuzumab amunaleukin) (RG 7813) (Roche), ticarcin UK (Camidanlumab tesirine) (ADC therapeutic Co., NHS-IL 2-EMD 5 (Merck 24)), NIZ985 (Noval Co., ltd.), MDNA (Medicenna Therapeutics (Alter Bioscience)), nagina (Cypraecox), nagin (Cypraecox Co., ltd.No. 2-Jilin), nagine (Cypraecox Bunge) and Nagine (Leu) and Nagine-201 (Cypraecox), nagine-201 (Cypraecox-201), nagine (Cypraecox-201) and Nagine (Cypraecox L-201 (Cypraecox L-35) THOR-809 (Sanofi)/Synthox), BNT151/153 (Biontech) of the family Baentaike, transCon IL-2 beta/gamma (Assendis Pharma), ILT-101 (Shi Weiya (Server)/ILT-101) and AM0015 (Lilly) of the family Baentai. Additional examples of IL-2 agonists are known in the art.

Complexes of common gamma chain family cytokines with antibodies or antibody fragments

Non-limiting examples of common gamma chain family cytokine receptor activators are complexes comprising a common gamma chain family cytokine (e.g., any of the common gamma chain family cytokines described herein) and an antibody or antigen-binding antibody fragment that specifically binds the common gamma chain family cytokine.

In some embodiments, a complex of a common gamma chain family cytokine and an antibody or antigen-binding antibody fragment that specifically binds the common gamma chain family cytokine may enhance the activity of the common gamma chain family cytokine and allow for CD8 ⁺ T cells and/or NK cells are expanded. In some embodiments, the complex has a longer circulating half-life than the free common gamma chain family cytokine.

In some embodiments, complexes can include soluble IL-2 (e.g., recombinant soluble human IL-2) or a functional fragment thereof, and an anti-IL-2 antibody or antigen-binding antibody fragment thereof. Non-limiting examples of complexes of soluble IL-2 and anti-IL-2 antibodies include soluble IL-2 complexed with anti-IL-2 antibody S4B6, JES6-5 or MAB602, respectively (see, e.g., tomala et al, J.Immunol.183:4904-4912, 2009; and Boyman et al, science 311, 2006).

In some embodiments, complexes can include soluble IL-4 (e.g., recombinant soluble human IL-4) and an anti-IL-4 antibody or antigen-binding antibody fragment thereof. Non-limiting examples of anti-IL-4 antibodies include those described in, for example, sato et al, J.Immunol.150:2717-2723, 1993 and Finkelman et al, J.Immunol.151:1235-1244, 1993.

In some embodiments, complexes can include soluble IL-7 (e.g., recombinant soluble human IL-7) and an anti-IL-7 antibody or antigen-binding antibody fragment thereof. Non-limiting examples of anti-IL-7 antibodies include those described in, for example, finkelman et al, J.Immunol.151:1235-1244, 1993, boyman et al, J.Immunol.180:7265-75, 2008.

In some embodiments of the complex, the common gamma chain family cytokine (or functional fragment thereof) and the antibody (or antigen-binding antibody fragment thereof) may be administered separately, and the complex between the common gamma chain family cytokine and the antibody or antigen-binding antibody fragment may be formed in vivo.

Additional examples of common gamma chain family cytokines and corresponding antibodies or antigen-binding antibody fragments that bind thereto are known in the art.

Exemplary methods comprising administering one or more agents that result in reduced TGF-beta receptor activation

Provided herein are methods of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

Also provided herein are methods of reducing naturally occurring and/or treatment-induced accumulation of senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

Also provided herein are methods of reducing the level of a marker of naturally occurring and/or treatment-induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more active agents that result in a TGF-beta receptorA reduced-dose agent. In some embodiments, the marker of naturally occurring and/or treatment-induced senescent cells is p21 ^CIP1 p21 and CD26. Additional markers of naturally occurring and/or treatment-induced senescent cells are described herein. Additional markers of naturally occurring and/or treatment-induced senescent cells are known in the art.

Also provided herein are methods of attenuating the activity of naturally occurring and/or treatment-induced senescent cells in a subject, comprising administering to the subject a therapeutically effective amount of one or more agents that result in attenuation of activation of a TGF- β receptor.

Also provided herein are methods of reducing the level and/or activity of one or more SASP factors derived from naturally occurring and/or therapeutically induced senescent cells in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of a TGF-beta receptor. In some embodiments, the senescent cells express an inflammatory feature, wherein the inflammatory feature is a SASP factor. In some embodiments, SASP factors include, but are not limited to, inflammatory cytokines (e.g., IL-1α, IL-1β, IL-6, IL-8, and TNF- α), growth factors (e.g., TGF- β, PDGF-AA, and insulin-like growth factor binding proteins (IGFBPs)), chemokines (e.g., CCL-2, CCL-20, CCL-7, CXCL-4, CXCL1, and CXCL-12), and matrix metalloproteinases (e.g., MMP-3 and MMP-9) that operate in a cell-autonomous manner to enhance aging (autocrine effects) and communicate with and alter the microenvironment (paracrine effects). In some embodiments, the method reduces the expression level or activity of one or more SASP factors. In some embodiments, the level of expression or activity of a SASP factor is determined using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the level of expression or activity of a SASP factor is determined using immunoblotting.

In some embodiments of any of the methods described herein, the subject has been previously diagnosed or identified as having an aging-related disease (e.g., any of the exemplary types of aging-related diseases or conditions described herein or known in the art) or an inflammatory disease (e.g., any of the exemplary types of aging-related diseases or conditions described herein or known in the art).

In some embodiments, the aging-related disorder is inflammatory aging-related.

In some embodiments, the aging-related disease is cancer (e.g., any of the exemplary types of cancers described herein or known in the art).

In some embodiments of any one of the methods described herein, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, crohn's disease, multiple sclerosis, guillain barre syndrome, psoriasis, grave's disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders and cognitive disorders associated with cancer treatment.

In some embodiments of these methods, the treatment-induced senescent cells are chemotherapy-induced senescent cells.

In some embodiments of these methods, for example, administration results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 5% to about 99% (or any subrange of this range described herein)) in the subject's amount of naturally occurring and/or treatment-induced senescent cells in the subject's target tissue (e.g., any of the exemplary types of target tissue described herein) compared to the amount of naturally occurring and/or treatment-induced senescent cells in the subject's target tissue prior to treatment.

In some embodiments of these methods, for example, administration results in a reduction (e.g., at least a reduction of 5%, at least a reduction of 10%, at least a reduction of 15%, at least a reduction of 20%, at least a reduction of 25%, at least a reduction of 30%, at least a reduction of 35%, at least a reduction of 40%, at least a reduction of 45%, at least a reduction of 50%, at least a reduction of 55%, at least a reduction of 60%, at least a reduction of 65%, at least a reduction of 70%, at least a reduction of 75%, at least a reduction of 80%, at least a reduction of 85%, at least a reduction of 90%, or at least a reduction of 95%, or a reduction of about 5% to about 99% (or any subrange of this range described herein)) in the subject compared to the naturally occurring and/or therapeutically induced accumulation of senescent cells in the subject prior to treatment or the naturally occurring and/or therapeutically induced senescent cells in a similar subject that did not receive the treatment.

In some embodiments of these methods, for example, administration results in a decrease (e.g., at least a decrease of 5%, at least a decrease of 10%, at least a decrease of 15%, at least a decrease of 20%, at least a decrease of 25%, at least a decrease of 30%, at least a decrease of 35%, at least a decrease of 40%, at least a decrease of 45%, at least a decrease of 50%, at least a decrease of 55%, at least a decrease of 60%, at least a decrease of 65%, at least a decrease of 70%, at least a decrease of 75%, at least a decrease of 80%, at least a decrease of 85%, at least a decrease of 90%, or at least a decrease of 95%, or a decrease of about 5% to about 99% (or any subrange of the ranges described herein)) in the level of one or more of the naturally occurring and/or therapeutically induced markers of senescent cells in the subject as compared to the level of the one or more of the naturally occurring and/or therapeutically induced markers of senescent cells in the subject prior to the treatment.

In some embodiments, the TGF-beta receptor is TGF-beta receptor II (TGF-beta RII).

In some embodiments, the TGF- β receptor is TGF- βRIII.

In some embodiments, at least one of the one or more agents that result in reduced activation of the TGF- β receptor is a soluble TGF- β receptor, an extracellular domain of a TGF- β receptor, an antibody that specifically binds to TGF- β, an antagonistic antibody that binds to TGF- β receptor, an agent that binds to LAP, or an agent that binds to a TGF- β/LAP complex. In some embodiments, one or more agents that result in reduced TGF- β receptor activation attenuate TGF- β receptor activation by binding to LAP or TGF- β/LAP complex. Non-limiting examples of agents that result in reduced TGF-beta receptor activation are described below.

Agents that result in reduced TGF-beta receptor activation

The methods provided herein include the use or administration of one or more agents that result in reduced TGF- β receptor activation. In some embodiments, the agent that results in reduced activation of the TGF- β receptor is a single chain chimeric polypeptide (e.g., any of the exemplary single chain chimeric polypeptides described herein), a multi-chain chimeric polypeptide (e.g., any of the exemplary multi-chain chimeric polypeptides described herein), a soluble TGF- β receptor, an extracellular domain of a TGF- β receptor, an antibody (or antibody fragment) that specifically binds TGF- β, an antagonistic antibody that binds TGF- β receptor, an agent that binds LAP, or an agent that binds TGF- β/LAP complex.

Exemplary Single chain chimeric Polypeptides

Non-limiting examples of agents that result in reduced TGF- β receptor activation are single chain chimeric polypeptides comprising: (i) a first target binding domain; (ii) A soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art); and (iii) a second target binding domain, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a ligand of a TGF- β receptor; or one or both of the first target binding domain and the second target binding domain is an antagonistic antigen binding domain that specifically binds to a TGF- β receptor. In some embodiments, the TGF- β receptor is TGF- βRII. In some embodiments, the TGF- β receptor is TGF- βRIII.

Some embodiments of any of the single-chain chimeric polypeptides described herein can further comprise one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located at the N-terminus and/or C-terminus thereof.

In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and/or the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble TGF- β receptor. Non-limiting examples of soluble TGF-beta receptors include soluble TGF-beta RI, soluble TGF-beta RII, soluble TGF-beta RIII, and soluble endoglin. Non-limiting sequences of exemplary soluble tgfbetarii are described herein.

Exemplary Multichain chimeric Polypeptides

A non-limiting example of an agent that results in reduced TGF- β receptor activation is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain; (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; (ii) A second target binding domain, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a ligand of a TGF- β receptor; or one or both of the first target binding domain and the second target binding domain is an antagonistic antigen binding domain that specifically binds to a TGF- β receptor.

In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and/or the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble TGF- β receptor. Non-limiting examples of soluble TGF-beta receptors include soluble TGF-beta RI, soluble TGF-beta RII, soluble TGF-beta RIII, and soluble endoglin.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the pair of affinity domains are the sushi domain and soluble IL-15 from the alpha chain of the human IL-15 receptor (il15rα). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, soluble IL-15 contains reduced or eliminated IL-15 activity mutations.

In some embodiments of any one of the multi-chain chimeric polypeptides described herein, the pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first domain or the second domain of a pair of affinity domains is a soluble common gamma chain family cytokine or an antigen binding domain that specifically binds to a common gamma chain family cytokine receptor.

Non-limiting examples of multi-chain chimeric polypeptides as agents that result in reduced activation of TGF-beta receptors are those described in the section entitled "exemplary B-, G-, I-, K-, L-, M-, N-, O-and P-type multi-chain chimeric polypeptides" herein.

Soluble TGF-beta receptor

In some embodiments, the one or more agents that result in reduced activation of the TGF- β receptor is a soluble TGF- β receptor. In some embodiments, the one or more agents that result in reduced activation of the TGF- β receptor is a soluble TGF- β receptor. Non-limiting examples of soluble TGF-beta receptors include soluble TGF-beta RI, soluble TGF-beta RII, soluble TGF-beta RIII, and soluble endoglin.

In some embodiments, the TGF-beta receptor is TGF-beta receptor II (TGF-beta RII). In some embodiments, the tgfβ receptor is tgfβriii.

TGF-beta RI, type I receptor, is a membrane-bound serine/threonine kinase that requires the presence of TGF-beta RII to bind TGF-beta. Tgfbetarii, type II receptor, is a membrane-bound serine/threonine kinase that binds TGF- β1 and TGF- β3 with high affinity and TGF- β2 with much lower affinity. In some embodiments, signal transduction requires the cytoplasmic domains of both tgfβri and tgfβrii. TGF-beta RIII, type III receptor, is a proteoglycan that exists in a membrane-bound and soluble form and binds TGF-beta 1, TGF-beta 2 and TGF-beta 3, but does not appear to be involved in signal transduction. Non-limiting examples of soluble TGF-beta RII sequences are described herein.

Antigen binding domains

In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each an antigen binding domain. In some embodiments of any of the single-or multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is a scFv or single domain antibody (e.g., a VHH or VNAR domain). In some embodiments of any of the single-or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is an antagonistic antigen binding domain that specifically binds to a TGF- β receptor. In some embodiments, an antagonistic antigen binding domain (e.g., any of the antigen binding domains described herein) can specifically bind to soluble tgfβri, soluble tgfβrii, soluble tgfβriii, or soluble endoglin.

In some embodiments, any of the methods described herein The antigen binding domain is BiTe, (scFv) ₂ Nanobody, nanobody-HAS, DART, tandAb, single chain diabody (scDiabody), single chain diabody-CH 3, scFv-CH-CL-scFv, HSAbody, single chain diabody-HSA or tandem scFv. Additional examples of antigen binding domains that can be used in any single or multi-chain chimeric polypeptide are known in the art.

In some embodiments, two or more polypeptides present in a single-chain or multi-chain chimeric polypeptide can be assembled (e.g., non-covalently assembled) to form any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), a VHH-scAb, a VHH-Fab, a dual scFab, a F (ab ') 2, a bifunctional antibody, an interchangeable mab, a DAF (two-in-one), a DAF (four-in-one), a DutaMab, a DT-IgG, a mortar-pestle type common light chain, a mortar-pestle type assembly, a charge pair, a Fab arm exchange, a SEEDbody, a LUZ-Y, fcab, a kappa lambda body, an orthogonal Fab, a DVD-IgG, an IgG (H) -scFv, a scFv- (H) IgG, an IgG (L) -scFv, a scFv- (L) IgG, an IgG (L), H) -Fv, igG (H) -V, V (H) -IgG, igG (L) -V, V (L) -IgG, KIH IgG-scFab, 2scFv-IgG, igG-2scFv, scFv4-Ig, zybody, DVI-IgG, diabody-CH 3, triad body, minibody, triBi minibody, scFv-CH3 KIH, fab-scFv, F (ab') 2-scFv2, scFv-KIH, fab-scFv-Fc, tetravalent HCAb, single chain diabody-Fc, tandem scFv-Fc, intracellular antibody, docking and locking (dock and lock), lmmTAC, igG-IgG conjugates, cov-X bodies, and scFv1-PEG-scFv2. See, e.g., spiess et al, mol. Immunol.67:95-106, 2015, which are incorporated herein in their entirety to describe these elements. Non-limiting examples of antigen binding fragments of antibodies include Fv fragments, fab fragments, F (ab') ₂ Fragments and Fab' fragments. Further examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, igG2, igG3, or IgG 4) (e.g., human or humanized IgG, such as antigen-binding fragments of human or humanized IgG1, igG2, igG3, or IgG 4); antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA 2) (e.g., human or humanizedIgA, e.g., an antigen binding fragment of human or humanized IgA1 or IgA 2); antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD); antigen binding fragments of IgE (e.g., antigen binding fragments of human or humanized IgE); or an antigen-binding fragment of IgM (e.g., an antigen-binding fragment of human or humanized IgM).

Agents that bind latency-associated peptides (LAP)

Non-limiting examples of agents that bind to latency-related peptides (LAP) are TGF- β1, thrombospondin-1 (TSP-1), integrin αvβ6 or KRFK peptides. In some embodiments, LAP binds TGF- β1 to form a latent complex, wherein LAP is presumed to act as a sequestering agent for active TGF- β1 (sequestering agent). In some embodiments, the LAP of the latent TGF-beta complex also interacts with thrombospondin-1 (TSP-1) as part of the bioactive complex. The formation of the TSP-1/LAP complex involves the activating sequence of TPS-1 (KRFK) and the sequence near the amino terminus of LAP (LSKL) conserved in TGF-beta 1-5. The interaction of LAP with TSP-1 through LSKL and KRFK sequences is important for thrombospondin-mediated latent TGF- β, as LSKL peptides can competitively inhibit latent TGF- β activation by TSP-1 or KRFK-containing peptides. In some embodiments, integrin αvβ6 has been shown to have high affinity for TGF- β1LAP and is involved in the activation of TGF- β1 latent complexes.

Agents that bind TGF-beta/LAP complexes

A non-limiting example of an agent that binds to the TFG- β/LAP complex is latent TGF- β binding protein (LTBP). In some embodiments, latent TGF-beta binding protein (LTBP) binds to the TFG-beta/LAP complex, forming a larger complex known as the Large Latent Complex (LLC). In some embodiments, the LTBP includes LTBP-1, LTBP-2, LTBP-3, and LTBP-4. In some embodiments, LTBP-1 forms disulfide-linked complexes with tgfβ propeptides (e.g., LAP) in the endoplasmic reticulum. In some embodiments, LTBP-4 binds only to TGF- β1, and thus, mutations in LTBP-4 may result in TGF- β related complications that are specific for tissues that are primarily involved in TGF- β1.

Application method

Some embodiments of the methods described herein include administering one or two or more doses (e.g., three or more doses, four or more doses, five or more doses, six or more doses, seven or more doses, eight or more doses, nine or more doses, or ten or more doses) of one or more agents that result in reduced TGF- β receptor activation in a subject. In some embodiments of these methods, any two consecutive doses of two or more doses are administered at intervals of about 1 week to about one year (e.g., from about 1 week to about 11 months, from about 1 week to about 10 months, from about 1 week to about 9 months, from about 1 week to about 8 months, from about 1 week to about 7 months, from about 1 week to about 6 months, from about 1 week to about 5 months, from about 1 week to about 4 months, from about 1 week to about 3 months, from about 1 week to about 2 months, from about 1 week to about 1 month, from about 1 week to about 3 weeks, from about 1 week to about 2 weeks, from about 2 weeks to about 12 months, from about 2 weeks to about 11 months, from about 2 weeks to about 10 months, from about 2 weeks to about 9 months, from about 2 weeks to about 8 months, from about 2 weeks to about 7 months, from about 2 weeks to about 6 months, from about 2 weeks to about 5 months, from about 2 weeks to about 4 months, from about 2 weeks to about 3 months, from about 2 weeks to about 2 months, from about 2 weeks to about 1 month, from about 2 weeks to about 1 week, from about 2 weeks to about 3 weeks, from about 3 weeks to about 12 months, from about 2 weeks to about 2 weeks, from about 2 weeks to about 11 months, from about 2 weeks to about 2 weeks, from about 3 months, from about 2 weeks to about 9 months. From about 3 weeks to about 11 months, from about 3 weeks to about 10 months, from about 3 weeks to about 9 months, from about 1 month to about 8 months, from about 3 weeks to about 7 months, from about 3 weeks to about 6 months, from about 3 weeks to about 5 months, from about 3 weeks to about 4 months, from about 3 weeks to about 3 months, from about 3 weeks to about 2 months, from about 3 weeks to about 1 month, from about 1 month to about 12 months, from about 1 month to about 11 months, from about 1 month to about 10 months, from about 1 month to about 9 months, from about 1 month to about 8 months, from about 1 month to about 7 months, from about 1 month to about 6 months, from about 1 month to about 5 months, from about 1 month to about 4 months, from about 1 month to about 3 months, from about 1 month to about 2 months, from about 2 months to about 12 months, from about 2 months to about 11 months, from about 2 months to about 10 months, from about 2 months to about 9 months, from about 2 months to about 8 months, from about 2 months to about 7 months, from about 2 months to about 6 months, from about 2 months to about 5 months, from about 2 months to about 4 months, from about 2 months to about 3 months, from about 3 months to about 12 months, from about 3 months to about 11 months, from about 3 months to about 10 months, from about 3 months to about 9 months, from about 3 months to about 8 months, from about 3 months to about 7 months, from about 3 months to about 6 months, from about 3 months to about 5 months, from about 3 months to about 4 months, from about 4 months to about 12 months, from about 4 months to about 11 months, from about 4 months to about 10 months, from about 4 months to about 9 months, from about 4 months to about 8 months, from about 4 months to about 7 months, from about 4 months to about 6 months to about 4 months, from about 4 months to about 4 months. From about 5 months to about 12 months, from about 5 months to about 11 months, from about 5 months to about 10 months, from about 7 months to about 11 months, from about 7 months to about 10 months, from about 7 months to about 9 months, from about 7 months to about 8 months, from about 8 months to about 12 months, from about 6 months to about 11 months, from about 6 months to about 10 months, from about 6 months to about 9 months, from about 6 months to about 8 months, from about 6 months to about 7 months, from about 7 months to about 12 months, from about 7 months to about 11 months, from about 7 months to about 10 months, from about 7 months to about 9 months, from about 7 months to about 8 months, from about 8 months to about 12 months, from about 8 months to about 10 months, from about 8 months to about 9 months, from about 9 months to about 11 months, from about 7 months to about 10 months, from about 8 months to about 12 months, from about 8 months to about 10 months, from about 10 months About 10 months to about 11 months, or about 11 months to about 12 months).

In some embodiments of any of the methods described herein, the one dose or two or more doses are administered by subcutaneous administration. In some embodiments of any of the methods described herein, the one or two or more doses are administered by intramuscular injection.

In some embodiments of any of the methods described herein, the two or more doses are administered for about 1 year to about 60 years (e.g., about 1 year to about 55 years, about 1 year to about 50 years, about 1 year to about 45 years, about 1 year to about 40 years, about 1 year to about 35 years, about 1 year to about 30 years, about 1 year to about 25 years, about 1 year to about 20 years, about 1 year to about 15 years, about 1 year to about 10 years, about 1 year to about 5 years, about 5 years to about 60 years, about 5 years to about 55 years, about 5 years to about 50 years, about 5 years to about 45 years, about about 5 years to about 40 years, about 5 years to about 35 years, about 5 years to about 30 years, about 5 years to about 25 years, about 5 years to about 20 years, about 5 years to about 15 years, about 5 years to about 10 years, about 10 years to about 60 years, about 10 years to about 55 years, about 10 years to about 50 years, about 10 years to about 45 years, about 10 years to about 40 years, about 10 years to about 35 years, about 10 years to about 30 years, about 10 years to about 25 years, about about 10 years to about 20 years, about 10 years to about 15 years, about 15 years to about 60 years, about 15 years to about 55 years, about 15 years to about 50 years, about 15 years to about 45 years, about 15 years to about 40 years, about 15 years to about 35 years, about 15 years to about 30 years, about 15 years to about 25 years, about 15 years to about 20 years, about 20 years to about 60 years, about 20 years to about 55 years, about 20 years to about 50 years, about 20 years to about 45 years, about 20 years to about 40 years, about 20 years to about 35 years, about 20 years to about 30 years, about 20 years to about 25 years, about 25 years to about 60 years, about 25 years to about 55 years, about 25 years to about 50 years, about 25 years to about 45 years, about 25 years to about 35 years, about 30 years to about 60 years, about 30 years to about 30 years, about 30 years to about 55 years, about 30 years to about 50 years, about 30 years to about 45 years, about 30 years to about 40 years, about 30 years to about 35 years, about 35 years to about 60 years, about 35 years to about 55 years, about 35 years to about 50 years, about 35 years to about 45 years, about 35 years to about 40 years, about 40 years to about 60 years, about 40 years to about 55 years, about 40 years to about 50 years, about 40 years to about 45 years, about 45 years to about 60 years, about 45 years to about 55 years, about 45 years to about 50 years, about 50 years to about 60 years, about 50 years to about 55 years, or about 55 years to about 60 years).

In some embodiments of these methods, each of the one or two or more doses is administered at a dose of about 0.01mg/kg of each agent that results in reduced TGF- β receptor activation to about 10mg/kg of each agent that results in reduced TGF- β receptor activation (e.g., about 0.01mg/kg to about 9mg/kg, about 0.01mg/kg to about 8mg/kg, about 0.01mg/kg to about 7mg/kg, about 0.01mg/kg to about 6mg/kg, about 0.01mg/kg to about 5mg/kg, about 0.01mg/kg to about 4mg/kg, about 0.01mg/kg to about 3mg/kg, about 0.01mg/kg to about 2mg/kg, about 0.01mg/kg to about 1mg/kg, about 0.01mg/kg to about 0.5mg/kg, about 0.01mg/kg to about 0.1mg/kg, about 0.01mg/kg to about 0.05mg/kg, about 0.05mg/kg to about 10mg/kg, about 0.05mg/kg to about 9mg/kg, about 0.05mg/kg to about 8mg/kg, about 0.05mg/kg to about 7mg, about 0.05mg/kg to about 6mg/kg, about 0.5mg/kg, about 0.05mg to about 1mg/kg, about 0.0.1 mg/kg to about 0.5mg/kg, about 0.01mg/kg to about 0.5mg/kg, about 0.05mg/kg to about 10mg/kg, about 0.05mg/kg to about 10mg/kg, about 0.1mg/kg to about 3mg/kg, about 0.1mg/kg to about 2mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.1mg/kg to about 0.5mg/kg, about 0.5mg/kg to about 10mg/kg, about 0.5mg/kg to about 9mg/kg, about 0.5mg/kg to about 8mg/kg, about 0.5mg/kg to about 7mg/kg, about 0.5mg/kg to about 6mg/kg, about 0.5mg/kg to about 5mg/kg, about 0.5mg/kg to about 4mg/kg, about 0.5mg/kg to about 3mg/kg, about 0.5mg/kg to about 2mg/kg, about 0.5mg/kg to about 1mg/kg to about 10mg/kg, about 1mg/kg to about 9mg/kg, about 1mg/kg to about 8mg/kg, about 1mg to about 7mg/kg, about 3mg to about 3mg/kg to about 6mg/kg, about 3mg to about 3mg/kg to about 2mg/kg, about 2mg to about 2mg/kg to about 3mg/kg, about 3mg to about 2mg/kg, about 3mg to about 2mg/kg to about 3mg/kg, about 5mg to about 4mg/kg, about 3mg/kg to about 4mg/kg, about 4mg/kg to about 10mg/kg, about 4mg/kg to about 9mg/kg, about 4mg/kg to about 8mg/kg, about 4mg/kg to about 7mg/kg, about 4mg/kg to about 6mg/kg, about 4mg/kg to about 5mg/kg, about 5mg/kg to about 10mg/kg, about 5mg/kg to about 9mg/kg, about 5mg/kg to about 8mg/kg, about 5mg/kg to about 7mg/kg, about 5mg/kg to about 6mg/kg, about 6mg/kg to about 10mg/kg, about 6mg/kg to about 9mg/kg, about 6mg/kg to about 8mg/kg, about 7mg/kg to about 10mg/kg, about 7mg/kg to about 9mg/kg, about 7mg/kg to about 8mg/kg, about 8mg/kg to about 10mg/kg, about 10mg to about 8mg/kg, about 10mg/kg to about 8mg/kg of β -receptor is administered.

In some embodiments of these methods, a single dose or first dose of one or more agents that result in reduced TGF- β receptor activation begins when the subject reaches at least 30 years old (e.g., at least 32 years old, 34 years old, 36 years old, 38 years old, 40 years old, 42 years old, 44 years old, 46 years old, 48 years old, 50 years old, 52 years old, 54 years old, 56 years old, 58 years old, 60 years old, 62 years old, 65 years old, 70 years old, 75 years old, or 80 years old).

In some embodiments of any one of the methods described herein, the subject is not diagnosed or identified as having an aging-related disease (e.g., any one of the aging-related diseases or conditions described herein or known in the art) or an inflammatory disease (e.g., any one of the aging-related diseases or conditions described herein or known in the art). In some embodiments of any of the methods described herein, the subject has not been previously treated with a chemotherapeutic agent (e.g., any of the chemotherapeutic agents described herein or known in the art). In some embodiments of any of the methods described herein, the subject has not been previously treated with a therapeutic agent that induces cellular senescence (e.g., any of the additional therapeutic agents described herein that induce cellular senescence).

Some embodiments of the methods described herein comprise administering one or two or more doses (e.g., three or more doses, four or more doses, five or more doses, six or more doses, seven or more doses, eight or more doses, nine or more doses, or ten or more doses) of one or more common gamma chain family cytokine receptor activators to a subject. In some embodiments of these methods, any two consecutive doses of two or more doses are administered at intervals of about 1 week to about one year (e.g., from about 1 week to about 11 months, from about 1 week to about 10 months, from about 1 week to about 9 months, from about 1 week to about 8 months, from about 1 week to about 7 months, from about 1 week to about 6 months, from about 1 week to about 5 months, from about 1 week to about 4 months, from about 1 week to about 3 months, from about 1 week to about 2 months, from about 1 week to about 1 month, from about 1 week to about 3 weeks, from about 1 week to about 2 weeks, from about 2 weeks to about 12 months, from about 2 weeks to about 11 months, from about 2 weeks to about 10 months, from about 2 weeks to about 9 months, from about 2 weeks to about 8 months, from about 2 weeks to about 7 months, from about 2 weeks to about 6 months, from about 2 weeks to about 5 months, from about 2 weeks to about 4 months, from about 2 weeks to about 3 months, from about 2 weeks to about 2 months, from about 2 weeks to about 1 month, from about 2 weeks to about 1 week, from about 2 weeks to about 3 weeks, from about 3 weeks to about 12 months, from about 2 weeks to about 2 weeks, from about 2 weeks to about 11 months, from about 2 weeks to about 2 weeks, from about 3 months, from about 2 weeks to about 9 months. From about 3 weeks to about 11 months, from about 3 weeks to about 10 months, from about 3 weeks to about 9 months, from about 1 month to about 8 months, from about 3 weeks to about 7 months, from about 3 weeks to about 6 months, from about 3 weeks to about 5 months, from about 3 weeks to about 4 months, from about 3 weeks to about 3 months, from about 3 weeks to about 2 months, from about 3 weeks to about 1 month, from about 1 month to about 12 months, from about 1 month to about 11 months, from about 1 month to about 10 months, from about 1 month to about 9 months, from about 1 month to about 8 months, from about 1 month to about 7 months, from about 1 month to about 6 months, from about 1 month to about 5 months, from about 1 month to about 4 months, from about 1 month to about 3 months, from about 1 month to about 2 months, from about 2 months to about 12 months, from about 2 months to about 11 months, from about 2 months to about 10 months, from about 2 months to about 9 months, from about 2 months to about 8 months, from about 2 months to about 7 months, from about 2 months to about 6 months, from about 2 months to about 5 months, from about 2 months to about 4 months, from about 2 months to about 3 months, from about 3 months to about 12 months, from about 3 months to about 11 months, from about 3 months to about 10 months, from about 3 months to about 9 months, from about 3 months to about 8 months, from about 3 months to about 7 months, from about 3 months to about 6 months, from about 3 months to about 5 months, from about 3 months to about 4 months, from about 4 months to about 12 months, from about 4 months to about 11 months, from about 4 months to about 10 months, from about 4 months to about 9 months, from about 4 months to about 8 months, from about 4 months to about 7 months, from about 4 months to about 6 months to about 4 months, from about 4 months to about 4 months. From about 5 months to about 12 months, from about 5 months to about 11 months, from about 5 months to about 10 months, from about 7 months to about 11 months, from about 7 months to about 10 months, from about 7 months to about 9 months, from about 7 months to about 8 months, from about 8 months to about 12 months, from about 6 months to about 11 months, from about 6 months to about 10 months, from about 6 months to about 9 months, from about 6 months to about 8 months, from about 6 months to about 7 months, from about 7 months to about 12 months, from about 7 months to about 11 months, from about 7 months to about 10 months, from about 7 months to about 9 months, from about 7 months to about 8 months, from about 8 months to about 12 months, from about 8 months to about 10 months, from about 8 months to about 9 months, from about 9 months to about 11 months, from about 7 months to about 10 months, from about 8 months to about 12 months, from about 8 months to about 10 months, from about 10 months About 10 months to about 11 months, or about 11 months to about 12 months).

In some embodiments of any of the methods described herein, the one dose or two or more doses are administered by subcutaneous administration. In some embodiments of any of the methods described herein, the one or two or more doses are administered by intramuscular injection.

In some embodiments of any of the methods described herein, the two or more doses are administered for about 1 year to about 60 years (e.g., about 1 year to about 55 years, about 1 year to about 50 years, about 1 year to about 45 years, about 1 year to about 40 years, about 1 year to about 35 years, about 1 year to about 30 years, about 1 year to about 25 years, about 1 year to about 20 years, about 1 year to about 15 years, about 1 year to about 10 years, about 1 year to about 5 years, about 5 years to about 60 years, about 5 years to about 55 years, about 5 years to about 50 years, about 5 years to about 45 years, about about 5 years to about 40 years, about 5 years to about 35 years, about 5 years to about 30 years, about 5 years to about 25 years, about 5 years to about 20 years, about 5 years to about 15 years, about 5 years to about 10 years, about 10 years to about 60 years, about 10 years to about 55 years, about 10 years to about 50 years, about 10 years to about 45 years, about 10 years to about 40 years, about 10 years to about 35 years, about 10 years to about 30 years, about 10 years to about 25 years, about about 10 years to about 20 years, about 10 years to about 15 years, about 15 years to about 60 years, about 15 years to about 55 years, about 15 years to about 50 years, about 15 years to about 45 years, about 15 years to about 40 years, about 15 years to about 35 years, about 15 years to about 30 years, about 15 years to about 25 years, about 15 years to about 20 years, about 20 years to about 60 years, about 20 years to about 55 years, about 20 years to about 50 years, about 20 years to about 45 years, about 20 years to about 40 years, about 20 years to about 35 years, about 20 years to about 30 years, about 20 years to about 25 years, about 25 years to about 60 years, about 25 years to about 55 years, about 25 years to about 50 years, about 25 years to about 45 years, about 25 years to about 35 years, about 30 years to about 60 years, about 30 years to about 30 years, about 30 years to about 55 years, about 30 years to about 50 years, about 30 years to about 45 years, about 30 years to about 40 years, about 30 years to about 35 years, about 35 years to about 60 years, about 35 years to about 55 years, about 35 years to about 50 years, about 35 years to about 45 years, about 35 years to about 40 years, about 40 years to about 60 years, about 40 years to about 55 years, about 40 years to about 50 years, about 40 years to about 45 years, about 45 years to about 60 years, about 45 years to about 55 years, about 45 years to about 50 years, about 50 years to about 60 years, about 50 years to about 55 years, or about 55 years to about 60 years).

In some embodiments of these methods, each dose of two or more doses of about 0.01mg/kg of each of the common gamma chain family cytokine receptor activators to about 10mg/kg of each of the common gamma chain family cytokine receptor activators (e.g., about 0.01mg/kg to about 9mg/kg, about 0.01mg/kg to about 8mg/kg, about 0.01mg/kg to about 7mg/kg, about 0.01mg/kg to about 6mg/kg, about 0.01mg/kg to about 5mg/kg, about 0.01mg/kg to about 4mg/kg, about 0.01mg/kg to about 3mg/kg, about 0.01mg/kg to about 2mg/kg, about 0.01mg/kg to about 1mg/kg, about 0.01mg/kg to about 0.5mg/kg, about 0.01mg/kg to about 1mg/kg, about 0.01mg/kg to about 7mg/kg, about 0.0.01 mg/kg to about 7mg/kg, about 0.1mg/kg to about 0.1mg/kg, about 0.01mg/kg to about 7mg/kg, about 0.1mg/kg to about 4mg/kg, about 0.01mg to about 2mg/kg, about 0.01mg to about 0.01mg/kg to about 1mg/kg, about 0.01mg to about 2mg/kg, about 0.01mg to about 1mg/kg to about 1.01 mg/kg, about 0.01mg to about 0.5mg/kg to about 2mg/kg, about 0.01mg to about 0.5mg/kg, about 0.1mg/kg to about 3mg/kg, about 0.1mg/kg to about 2mg/kg, about 0.1mg/kg to about 1mg/kg, about 0.1mg/kg to about 0.5mg/kg, about 0.5mg/kg to about 10mg/kg, about 0.5mg/kg to about 9mg/kg, about 0.5mg/kg to about 8mg/kg, about 0.5mg/kg to about 7mg/kg, about 0.5mg/kg to about 6mg/kg, about 0.5mg/kg to about 5mg/kg, about 0.5mg/kg to about 4mg/kg, about 0.5mg/kg to about 3mg/kg, about 0.5mg/kg to about 2mg/kg, about 0.5mg/kg to about 1mg/kg to about 10mg/kg, about 1mg/kg to about 9mg/kg, about 1mg/kg to about 8mg/kg, about 1mg to about 7mg/kg, about 3mg to about 3mg/kg to about 6mg/kg, about 3mg to about 3mg/kg to about 2mg/kg, about 2mg to about 2mg/kg to about 3mg/kg, about 3mg to about 2mg/kg, about 3mg to about 2mg/kg to about 3mg/kg, about 5mg to about 4mg/kg, about 3mg/kg to about 4mg/kg, about 4mg/kg to about 10mg/kg, about 4mg/kg to about 9mg/kg, about 4mg/kg to about 8mg/kg, about 4mg/kg to about 7mg/kg, about 4mg/kg to about 6mg/kg, about 4mg/kg to about 5mg/kg, about 5mg/kg to about 10mg/kg, about 5mg/kg to about 9mg/kg, about 5mg/kg to about 8mg/kg, about 5mg/kg to about 7mg/kg, about 5mg/kg to about 6mg/kg, about 6mg/kg to about 10mg/kg, about 6mg/kg to about 9mg/kg, about 6mg/kg to about 8mg/kg, about 6mg/kg to about 7mg/kg, about 7mg/kg to about 10mg/kg, about 7mg/kg to about 9mg/kg, about 7mg/kg to about 8mg/kg, about 8mg/kg to about 10mg/kg, about 10mg/kg to about 8mg/kg or about 10mg/kg of the co-activated receptor.

In some embodiments of these methods, the single dose or first dose of one or more common gamma chain family cytokine receptor activators begins when the subject reaches at least 30 years old (e.g., at least 32 years old, 34 years old, 36 years old, 38 years old, 40 years old, 42 years old, 44 years old, 46 years old, 48 years old, 50 years old, 52 years old, 54 years old, 56 years old, 58 years old, 60 years old, 62 years old, 65 years old, 70 years old, 75 years old, or 80 years old).

In some embodiments of any one of the methods described herein, the subject is not diagnosed or identified as having an aging-related disease (e.g., any one of the aging-related diseases or conditions described herein or known in the art) or an inflammatory disease (e.g., any one of the aging-related diseases or conditions described herein or known in the art). In some embodiments of any of the methods described herein, the subject has not been previously treated with a chemotherapeutic agent (e.g., any of the chemotherapeutic agents described herein or known in the art). In some embodiments of any of the methods described herein, the subject has not been previously treated with a therapeutic agent that induces cellular senescence (e.g., any of the additional therapeutic agents described herein that induce cellular senescence).

Examples

The invention is further described in the following examples, which do not limit the scope of the invention as set forth in the claims.

Example 1: immunostimulatory C57BL/6 mice using a multi-chain polypeptide

Materials and methods

Exemplary multi-chain polypeptides (type a multi-chain polypeptides described herein) are generated that include a first polypeptide that is a soluble fusion of two tgfbetarii domains, a human tissue factor 219 fragment, and human IL-15, and a second polypeptide that is a soluble fusion of two tgfbetarii domains and a human IL-15 ra chain sushi domain.

Results

Immunostimulation of C57BL/6 mice

Wild-type C57BL/6 mice were treated subcutaneously with control PBS solution or with the multi-chain polypeptide at doses of 0.3mg/kg, 1mg/kg, 3mg/kg or 10mg/kg, respectively. Four days after treatment, spleen weight and the percentage of various immune cell types present in the spleen were assessed. Specifically, single spleen cell suspensions were generated and stained with fluorochrome-conjugated antibodies (including anti-CD 4, anti-CD 8, anti-NK 1.1 and anti-CD 19). Assessment of CD4 present in spleen of mice treated with control solution or multi-chain polypeptide by flow cytometry ⁺ T cells, CD8 ⁺ T cells, natural Killer (NK) cells and CD19 ⁺ Percentage of B cells. As shown in fig. 1A, spleen weight of mice treated with the multi-chain polypeptide increased with increasing doses of the multi-chain polypeptide. Furthermore, the spleen weights of mice treated with 1mg/kg, 3mg/kg and 10mg/kg of the multi-chain polypeptide were significantly higher than those of control solution treated mice, respectively. As shown in FIG. 1B, CD8 in spleen of mice treated with the multi-chain polypeptide ⁺ The percentage of both T cells and NK cells increases with increasing doses of the multi-chain polypeptide. Specifically, CD8 in mice treated with 0.3mg/kg, 3mg/kg and 10mg/kg of the multi-chain polypeptide ⁺ The percentage of T cells was higher than that of the control treated mice, and the percentage of NK cells in mice treated with 0.3mg/kg, 1mg/kg, 3mg/kg and 10mg/kg of the multi-chain polypeptide was higher than that of the control treated mice. These results indicate that exemplary multi-chain polypeptides are capable of stimulating immune cells, particularly CD8, in the spleen ⁺ T cells and NK cells.

Pharmacokinetics of

The pharmacokinetics of exemplary multi-chain polypeptides in wild-type C57BL/6 mice were assessed. Mice were treated subcutaneously with a 3mg/kg dose of the multi-chain polypeptide. Blood was collected via the tail vein at various time points and serum was prepared. The concentration of the multi-chain polypeptide in the serum was determined by ELISA. Briefly, the multi-chain polypeptides are captured using anti-human tissue factor antibodies and detected using biotinylated anti-human tgfβ receptor, peroxidase-conjugated streptavidin, and ABTS substrate. The results indicate that the half-life of the exemplary multi-chain polypeptide is 12.66 hours.

Immunostimulation of C57BL/6 mice over time

To assess the immunostimulatory effect of the multi-chain polypeptides over time, mice were treated with a single dose of 3mg/kg multi-chain polypeptide, and immediately after treatment and at 16, 24, 48, 72 and 92 hours after treatment, spleen weight and percent immune cell types present in the spleen were assessed using the techniques described above. As shown in fig. 2A, spleen weight of the multi-chain polypeptide treated mice increased at 48 hours post-treatment and continued to increase for the next 44 hours. In addition, as shown in FIG. 2B, CD8 was found in the spleen of mice treated with the multi-chain polypeptide ⁺ The percentages of T cells and NK cells increased at 48 hours after treatment and continued to increase during the next 44 hours. These results further demonstrate that exemplary multi-chain polypeptides are capable of stimulating immune cells, particularly CD8, in the spleen over time ⁺ T cells and NK cells.

CD8 ⁺ Proliferation enhancement of T cells and NK cells and granzyme B expression enhancement thereof

To assess the immune cell proliferation and cytotoxic potential induced by the multi-chain polypeptide, mice were treated with a single dose of 3mg/kg multi-chain polypeptide, and the spleens of these mice were assessed immediately after treatment and at 16, 24, 48, 72 and 92 hours after treatment. Briefly, a single spleen cell suspension was generated and stained with fluorochrome-conjugated antibodies to different cell types, including anti-CD 4, anti-CD 8, anti-NK 1.1 and anti-CD 19, as well as anti-Ki 67 antibodies (i.e., cell proliferation markers) and anti-granzyme B antibodies (i.e., cytotoxicity markers). The Ki67 and granzyme B Mean Fluorescence Intensities (MFI) of each immune cell type were analyzed by flow cytometry. As shown in FIGS. 3A and 3B, immediately after the treatment (0 hours) The expression of Ki67 and granzyme B of NK cells was increased at 24 hours and at each time point subsequently assessed. Furthermore, CD8 compared with immediately after the treatment (0 hours) ⁺ The expression of Ki67 and granzyme B by T cells increased at 48 hours and at each time point assessed subsequently. Thus, a single dose of a multi-chain polypeptide results in CD8 ⁺ Proliferation of T cells and NK cells is maintained for up to at least 4 days after treatment.

These results indicate that the multi-chain polypeptide not only caused CD8 in the spleen ⁺ The number of T cells and NK cells increases and the proliferation and cytotoxicity of these cells increases.

Cytotoxicity against tumor cells

Next, cytotoxicity of spleen cells activated by the multi-chain polypeptide against tumor cells was assessed in C57BL/6 mice. Mouse Moloney (Moloney) leukemia cells (Yac-1) were labeled with CellTrace Violet (CellTrace Violet) and used as tumor target cells. C57BL/6 mice were treated with 3mg/kg single dose of multi-chain polypeptide, and spleen cells were prepared and used as effector cells at multiple time points later. Target tumor cells were mixed with effector cells at a ratio of 10:1 effector to target (E: T) and incubated at 37℃for 20 hours. Target cell viability was assessed by analyzing ionogenic Propidium Iodide (PI) positive Yac-1 cells using flow cytometry. Percent inhibition of Yac-1 tumors was calculated using the formula:

Percent Yac-1 tumor inhibition = (number of Yac-1 viable cells in 1-experiment sample/number of Yac-1 viable cells in sample without splenocytes) x100

As shown in fig. 4, spleen cells of mice after 24 hours or more of treatment with the multi-chain polypeptide showed enhanced cytotoxicity against Yac-1 cells compared to spleen cells of untreated mice.

Example 2: immunostimulation of C57BL/6 mice using a high fat feed based model of type 2 diabetes mice

Materials and methods

TGFRt15-TGFRs are multi-chain chimeric polypeptides (type A multi-chain chimeric polypeptides described herein) that include two TGF-beta binding domains that are soluble human TGF-beta RII dimers (aa 24-159). 21t15-TGFRs are multi-chain chimeric polypeptides (type A multi-chain chimeric polypeptides described herein) comprising IL-21 and a TGF-beta binding domain. 2t2 is a chimeric polypeptide comprising two IL-2 polypeptides (chimeric polypeptides of type B described herein).

Results

To evaluate the effect of TGFRt15-TGFRs, 2t2 and 21t15-TGFRs on treatment of type 2 diabetes, a high fat diet based mouse model of type 2 diabetes (B6.129P2-ApoE from jackson laboratories ^tm1Unc /J). Mice were fed control feed or high fat feed for 11 weeks. The subpopulations of mice fed high fat diet were also treated with TGFRt15-TGFRs, 2t2 or 21t 15-TGFRs. Mice fed control diet, high fat diet and mice fed high fat diet and treated with TGFRt15-TGFRs, 2t2 or 21t15-TGFRs were evaluated 4 days after treatment. Briefly, a single spleen cell suspension was generated and stained with fluorochrome-conjugated antibodies (including anti-CD 4, anti-CD 8, anti-NK 1.1 and anti-CD 19). Assessment of CD4 present in spleens of mice of each group using flow cytometry ⁺ T cells, CD8 ⁺ T cells, natural Killer (NK) cells and CD19 ⁺ Percentage of B cells.

As shown in fig. 5A, the percentage of NK cells in PBMCs increased significantly after treatment with TGFRt15-TGFRs or 2t2, but not after 21t15-TGFRs treatment, compared to untreated mice. In addition, CD8 in PBMC after TGFRt15-TGFRs, 2t2 or 21t15-TGFRs treatment compared to untreated mice ⁺ The T cell percentage increases significantly. In addition, anti-Ki 67 antibodies were also used to evaluate CD4 in PBMCs ⁺ T cells, CD8 ⁺ T cells, natural Killer (NK) cells and CD19 ⁺ Proliferation of B cells. As shown in FIG. 5B, after treatment with TGFRt15-TGFRs, proliferated NK cells, CD4 ⁺ T cells and CD8 ⁺ The number of T cells increased significantly but not after treatment with 2T2 or 21T 15-TGFRs.

To examine the effect of TGFRt15-TGFRs, 2t2 and 21t15-TGFRs on animal skin and hair appearance and texture, mice were fed control feed or high fat feed for 7 weeks, and subpopulations of mice fed high fat feed were also treated with TGFRt15-TGFRs, 2t2 or 21t 15-TGFRs. One week after treatment, the appearance of the mice was assessed. Mice fed high fat diet and untreated, or mice fed high fat diet and treated with 21t15-TGFRs exhibited untidy hair and crumpled hair, and increased white hair/hair loss compared to mice fed control diet (fig. 6A, 6B, and 6E). Surprisingly, mice fed high fat diet and receiving TGFRt15-TGFRs or 2t2 treated appeared to be hair tidy and healthier (less white hair/hair loss) compared to mice fed high fat diet and not receiving TGFRt15-TGFRs or 2t2 treated (fig. 6B). Specifically, TGFRt15-TGFRs or 2t2 treated mice showed excellent skin and hair appearance and texture compared to control mice. These results indicate that treatment with TGFRt15-TGFRs or 2t2 can improve the appearance and texture of mammalian skin and hair.

Next, mice were fed control feed or high fat feed for 9 weeks, and a subset of mice fed high fat feed were treated with TGFRt15-TGFRs, 2t2 or 21t 15-TGFRs. Four days after treatment, the fasting body weight of each group of mice was measured. Mice fed high fat diet and untreated as well as mice fed high fat diet and treated with 21t15-TGFRs had significantly increased fasting body weight compared to mice fed control diet. However, the fasting body weight of mice fed high fat diet and treated with TGFRt15-TGFRs or 2t2 was reduced compared to the other two high fat diet groups described above. The fasting body weight of the mice at the end of the study (week 9) is shown in figure 7.

To evaluate fasting glucose levels in each group of mice, the mice were fed control feed or high fat feed and either untreated or treated with TGFRt15-TGFRs, 2t2 or 21t15-TGFRs on days 44, 59 and 73. Fasting blood glucose was measured in each group of mice 4 days after treatment. As shown in fig. 8, the fasting blood glucose levels were significantly reduced in mice fed high fat diet and treated with 2t2 (red line) compared to mice fed high fat diet but untreated (yellow line) after the second and third doses (on day 59 and 73, respectively). The fasting blood glucose levels of mice fed high fat diet and treated with TGFRt15-TGFRs remained constant (green line), while the fasting blood glucose levels of mice fed high fat diet and treated with 21t15-TGFRs increased (blue line).

Example 3: chemotherapy-induced expression of NK ligands by senescent B16F10 melanoma cells

Materials and methods

Cell senescence of B16F10 melanoma cells was induced by treatment of the cells with docetaxel (7.5 μm, sigma) for 3 days followed by 4 days recovery in complete medium. Cell senescence is known by staining cells with senescence-associated beta-galactosidase (SA beta-gal). Briefly, B16F10 control cells and senescent cells (B16F 10-SNC) were washed once with PBS and fixed with 0.5% glutaraldehyde (PBS (pH 7.2)) for 30 min. Cells were treated with X-gal solution (1 mg/mL of X-gal, 0.12mM K at 37 ℃C ₃ Fe[CN] ₆ K at 0.12mM ₄ Fe[CN] ₆ And 1mM MgCl ₂ pH 6.0) in PBS and imaged using a Nikon (Nikon) optical microscope.

Results

Chemotherapy as described above was used to induce B16F10 melanoma cells to develop cellular senescence. As shown in FIG. 9A, chemotherapy-induced senescent B16F10 cells (B16F 10-SNC) were positive for SA beta-gal staining, whereas control B16F10 cells were not stained. Next, expression of senescence genes was analyzed using RT-qPCR, wherein RNA was isolated on day 0 or after induction of senescence on days 4, 8, 12 and 16, respectively. Expression levels were normalized to control B16F10 cells. As shown in FIGS. 9B-9D, expression of p21, IL6 and DPP4 was up-regulated in RNA isolated from senescent cells during the course of the experiment. In addition, as shown in fig. 9E and 9F, expression of RATE1E and ULBP1 (NK-activating receptor NKG2D ligand) was also induced in senescent cells, with the highest expression level occurring at day 16. These results indicate that chemotherapy-induced senescent B16F10 cells underwent greater cytotoxicity of activated NK cells than control B16F10 cells.

Acquisition of stem cell characteristics of chemotherapy-induced senescent B16F10 melanoma cells

To examine whether chemotherapy-induced senescent B16F10 melanoma cells have stem cell characteristics, colony formation experiments were performed. Simple and easyFor example, 1000 cells were seeded per well on a six well plate and the medium was changed every three days. As shown in fig. 10A (images were taken at 100 x magnification), after 5 weeks of culture, senescent cells were able to form colonies. To assess the expression of stem cell markers of colonies, RNA was isolated from the colonies and the expression of Oct4 and Notch4 mRNA was determined by RT-qPCR. Chemotherapy-induced senescent B16F10 melanoma cells showed upregulation of the cancer stem cell markers Oct4 and Notch4 compared to control B16F10 cells (fig. 10B and 10C). Furthermore, the expression of stem cell markers CD44, CD24 and CD133 on the cell surface was assessed by staining with antibodies against CD44, CD24 and CD133 followed by flow cytometry. As shown in fig. 10D-10F, a double positive population (CD 44) in chemotherapy-induced senescent stem cells (B16F 10-SNC-CSC) ⁺ CD24 ⁺ 、CD44 ⁺ CD133 ⁺ And CD24 ⁺ CD133 ⁺ ) Increased compared to control B16F 10.

Melanoma cells with stem cell characteristics that are chemotherapy-induced aging (CIS) are more "migratory" and "invasive" than control B16F10 cells "

Migration characteristics of melanoma cells (B16F 10-SNC-CSC) with stem cell characteristics were analyzed by chemotherapy-induced aging (CIS) using a migration assay. Briefly, control B16F10 cells and B16F10-SNC-CSC cells were seeded on six well plates and injured with p20 pipette tips. The movement of the cells was imaged after 0, 12 and 24 hours. As shown in fig. 11A, in the in vitro migration analysis, the mobility of melanoma cells (B16F 10-SNC-CSC) with stem cell characteristics of chemotherapy-induced aging (CIS) was greater than that of control B16F10 cells.

Next, the invasion profile of chemotherapy-induced senescent cells with stem cell profile (B16F 10-SNC-CSC) was analyzed using invasion assay. Invasive analysis was performed on a Matrigel (Matrigel) coated 24-well plug-in co-culture cell. In brief, 0.5x10 ⁶ The individual control B16F10 cells and B16F10-SNC-CSC cells were inoculated in serum-free medium in the upper chamber, while the lower chamber was filled with medium supplemented with 10% FBS. After 16 hours incubation, the filter was removedCells on the upper surface, while cells under the filter were fixed and stained with 0.02% crystalline violet solution. The number of cells in the three fields of view was counted at 100 times magnification. As shown in fig. 11B and 11C, chemotherapy-induced senescent cells with stem cell characteristics were more invasive than control B16F10 cells when invading matrigel coats. These results indicate that chemotherapy-induced senescent B16F10 tumor cells are able to regain their proliferative capacity, acquire stem cell characteristics, and have enhanced migratory capacity and invasiveness for metastasis.

Cytotoxic activity of murine NK cells against chemotherapy-induced senescent cells with stem cell properties

To expand NK cells in vivo, TGFRt15-TGFRs (10 mg/kg) was subcutaneously injected into C57BL/6 mice for 4 days. Spleens were obtained from these mice and NK cells were purified using MACS Miltenyi column. Purified NK cells were then amplified in vitro using 2t2 (fig. 12A).

To assess cytotoxicity of expanded NK cells, chemotherapy-induced senescent stem cells (B16F 10-SNC-CSC) or control B16F10 cells were labeled with CellTrace violet and incubated with activated 2T2 mouse NK cells isolated from spleens of C57BL/6 mice maintained for 4 days with 10mg/kg TGFRt15-TGFRs in vitro at various E: T ratios for 16 hours. B16F10-SNC-CSC and control B16F10 cells were treated with trypsin, washed and resuspended in complete medium containing Propidium Iodide (PI) solution and cytotoxicity was known by flow cytometry. As shown in fig. 12B, NK cells were more effective in killing chemotherapy-induced senescent cells with stem cell properties (B16F 10-SNC-CSC) than control B16F10 cells.

Combined treatment of melanoma mouse models

The effect of TGFRt15-TGFRs on treatment of melanoma in the murine melanoma model was evaluated. Briefly, 5×10 ⁵ Individual B16F10 cells were subcutaneously injected into C57BL/6 mice. When the tumor volume reaches about 100mm ³ At this time, mice were treated every three days with docetaxel (chemotherapy) (5 mg/kg) or TA99 (200. Mu.g) as a single agent or in combination, and TGFRt15-TGFRs (3 mg-kg) once (fig. 13A). Mice that received normal saline, docetaxel (chemotherapy) alone/TA 99, or TGFRt15-TGFRs alone were used as controls. Five mice were tested per experimental and control group. Tumor volumes were measured every three days. As shown in fig. 13B and 13C, TGFRt15-TGFRs combined chemotherapy or TA99 slowed tumor progression in the syngeneic melanoma mouse model compared to saline-treated mice or mice treated with chemotherapy alone or TA 99.

Example 4: chemotherapy agents induce senescence in human pancreatic cell line SW1990

Materials and methods

Beta-galactosidase staining: chemotherapy-induced senescence was confirmed by standard β -galactosidase staining (pH 6.0) using a commercially available kit (cell signaling technique (Cell Signaling Technology)) according to manufacturer's instructions. The next day, the staining solution was removed and the cells were washed with phosphate buffered saline and 70% glycerol was added to the wells. Beta-galactosidase positive cells will be stained blue, while untreated control cells will not be stained.

Flow cytometry: one million control cells and senescent cells were obtained and stained according to the manufacturer's instructions using commercially available antibodies to stem cell surface markers, such as anti-CD 44 and anti-CD 24 antibodies (biotech (BioLegend)). The cells were then washed and analyzed using a BD Celesta flow cytometer. Cells exhibiting stem cell-like properties are double positive for CD44 and CD 24.

Analysis of gene expression: one million control cells and senescent cells were obtained and lysed using Trizol (sameiser), followed by RNA purification using RNA isolation kit (Qiagen). RNA was quantified and converted to cDNA using the cDNA Quantitect kit of Kanji. The cDNA was then used as a template for standard Taqman gene expression analysis (Siemens' Feier) for quantifying the relative abundance of senescence, stem cell markers, and NK ligands.

NK cell cytotoxicity assay: NK cells were isolated from healthy human donors (n=2) using a commercially available NK isolation kit (Stem Cell) and activated overnight using cytokine fusion molecules 18t15-12s (100 nM). The next day, NK cells were washed to remove cytokine molecules and mixed with CellTrace violet labeled untreated control tumor cells or chemotherapy-induced senescent tumor cells at a 4:1 E:T ratio for 20 hours. The next day, cells were digested with trypsin and the total content of each well was analyzed using flow cytometry and the percent inhibition of cells was analyzed.

Results

Human pancreatic tumor cell lines SW1990 were induced to age by treatment with 2.5 μm and 6.25 μm of the chemotherapeutic drugs albumin paclitaxel (Celgene) and gemcitabine (Sigma Aldrich), respectively, for 3 days. Untreated SW1990 cells served as control. After 3 days the medium was changed and the cells were allowed to stand in the medium for 4 days. As shown in fig. 14, the beta-galactosidase staining of the aged cells treated with the chemotherapeutic drug was positive (blue), while the control cells were not stained. Aging and stem cell marker expression of senescent and control cells were assessed 4, 11 and 22 days post-treatment. As shown in fig. 14, the double positive staining of senescent cells for CD44 and CD24 increased over time as compared to control cells. Furthermore, on day 0 and on days 2, 4 and 24 post-treatment, expression of senescence markers (including DPP4, IL6 and p 21), stem cell markers (including Oct3/4, CD24 and CD 44) and NK ligands (including connexin and MICA) of chemotherapy-induced senescent SW1990 cells was also analyzed using the gene expression assay described above. As shown in fig. 15, the expression of all the mentioned markers increased over time.

Cytotoxicity of in vitro activated human NK cells

To assess cytotoxicity of in vitro activated human NK cells (treated with 18t15-12 s) aging of human pancreatic tumor cell line SW1990 was induced by treatment with 2.5. Mu.M and 6.25. Mu.M of the chemotherapeutic drugs Albumin paclitaxel and gemcitabine (Sigma Aldrich) respectively for 3 days. Untreated SW1990 cells served as control. After 3 days the medium was changed and the cells were allowed to stand in the medium for 30 days. The medium was changed every 4 days. Activated NK cells were obtained and evaluated for cytotoxicity against chemotherapy-induced senescent tumor cells and untreated control tumor cells using the above NK cell cytotoxicity assays. As shown in fig. 16, activated NK cells showed increased cytotoxicity to both control SW1990 cells (SW 1990) and senescent SW1990 cells (SW 1990 s).

Example 5: IL-12/IL-15RαSu Production of DNA constructs

In one non-limiting embodiment, IL-12/IL-15RαSu DNA constructs were generated (FIG. 17). Human IL-12 subunit sequences, human IL-15RαSu sequences, human IL-15 sequences, human tissue factor 219 sequences and human IL-18 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi (Genewiz). A DNA construct was made, with a GS (3) linker connecting IL-12 subunit β (p 40) to IL-12 subunit α (p 35) to generate a single-stranded form of IL-12, and then directly connecting the IL-12 sequence to the IL-15RαSu sequence. The final IL-12/IL-15RαSu DNA construct sequence was synthesized by Jin Weizhi.

The nucleic acid sequence (including the signal peptide sequence) of the IL12/IL-15RαSu construct is shown below (SEQ ID NO: 181):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

(human IL-12 subunit beta (p 40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC

(Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human IL-12 subunit alpha (p 35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

Example 6: production of IL-18/TF/IL-15 DNA constructs

In one non-limiting example, the IL-18/TF/IL-15 construct is made as follows (FIG. 18): the IL-18 sequence is linked to the N-terminal coding region of tissue factor 219, and the IL-18/TF construct is further linked to the N-terminal coding region of IL-15. The nucleic acid sequence (including the leader sequence) of the IL-18/TF/IL-15 construct synthesized by Jin Weizhi is shown below (SEQ ID NO: 177):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC

(human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Example 7: secretion of IL-12/IL-15RαSu and IL-18/TF/IL-15 fusion proteins

IL-12/IL-15RαSu and IL-18/TF/IL-15 DNA constructs were cloned into pMSGV-1 modified retroviral expression vectors (as described in Hughes, hum Gene Ther 16:457-72, 2005, which is incorporated herein by reference) and the expression vectors transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allowed the formation and secretion of soluble IL-18/TF/IL-15:IL-12/IL-15RαSu protein complexes (designated 18t15-12s; FIGS. 19 and 20). The 18t15-12s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography to give a soluble (non-aggregate) protein complex consisting of IL-12/IL-15rαsu and IL-18/TF/IL-15 fusion proteins.

The amino acid sequence (including the signal peptide sequence) of the IL12/IL-15RαSu fusion protein is shown below (SEQ ID NO: 180):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-12 subunit beta (p 40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

(Joint)

GGGGSGGGGSGGGGS

(human IL-12 subunit alpha (p 35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIR

The amino acid sequence (including the signal peptide sequence) of the IL-18/TF/IL-15 fusion protein is shown below (SEQ ID NO: 176):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

Example 8: purification of 18t15-12s by immunoaffinity chromatography

Attaching anti-TF antibody affinity columns to general electric medical group (GE Healthcare) ^TM ) AKTA Avant protein purification system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Cell culture collections of 18t15-12s were adjusted to pH 7.4 with 1M Tris base and loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). Absorbance at 280nm was collected and the samples were then neutralized to pH 7.5-8.0 by adding 1M Tris base. Then using a molecular weight cut-off value of 30kDa The filter was centrifuged and the neutralized sample was buffer exchanged in PBS. FIG. 21 shows that the 18t15-12s complex binds to an anti-TF antibody affinity column where TF is the 18t15-12s binding partner. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 10 column volumes of PBS, 0.05% sodium azide, and stored at 2-8 ℃.

Example 9: size exclusion chromatography for 18t15-12s

General electric medical group200 The Increase 10/300 GL gel filtration column was connected to the general electric medical community AKTA ^TM Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8mL/min. 200. Mu.L of 1mg/mL of the 18t15-12s complex was injected onto the column using a capillary loop. The injections were followed by 1.25 column volumes of PBS. SEC chromatography is shown in figure 22. There is a major 18t15-12s protein peak and a smaller high molecular weight peak, probably due to the different degree of glycosylation of 18t15-12s dimers or aggregates.

Example 10: SDS-PAGE of 18t15-12s

To determine purity and protein molecular weight, purified 18t15-12s protein samples were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel was treated with InstantBuue ^TM Dyeing for about 30min, and decolorizing in purified water overnight. FIG. 23 shows an exemplary SDS gel of affinity purification of anti-TF antibodies at 18t15-12s with bands at expected molecular weights (66 kDa and 56 kDa).

Example 11: 18t15-12s glycosylation in CHO-K1 cells

18t15-12s glycosylation in CHO-K1 cells was confirmed according to the manufacturer's instructions using the protein deglycosylation mixture II kit (New England Biolabs (New England Biolabs)). FIG. 24 shows exemplary SDS PAGE of deglycosylated and non-deglycosylated 18t15-12 s. Deglycosylation reduces the molecular weight of 18t15-12s, as shown in FIG. 24, lane 4.

Example 12: quantification of recombinant proteins of the 18t15-12s Complex

18t15-12s complexes were detected and quantified using standard sandwich ELISA methods (FIGS. 25-28). The anti-human tissue factor antibody served as a capture antibody and the biotinylated anti-human IL-12, IL-15 or IL-18 antibodies (BAF 219, BAM 247, D045-6, all from Andi organisms) served as detection antibodies. The tissue factor in the purified 18t15-12s protein complex was also detected using an anti-human tissue factor capture antibody (I43) and an anti-human tissue factor antibody detection antibody. The I43/anti-TF antibody ELISA was compared to purified tissue factor at similar concentrations.

Example 13: immunostimulatory capacity of the 18t15-12s Complex

To assess the IL-15 immunostimulatory activity of the 18t15-12s complex, increasing concentrations of 18t15-12s were added to 200. Mu.L of 32D beta cells (104 cells/well) in IMDM:10% FBS medium. 32D beta cells were incubated at 37℃for 3 days. On the fourth day, WST-1 proliferation reagent (10. Mu.L/well) was added, and after 4 hours, absorbance was measured at 450nm to determine cell proliferation based on cleavage of WST-1 into soluble formazan dye. The biological activity of human recombinant IL-15 was evaluated as a positive control. As shown in FIG. 29, 18t15-12s exhibited IL-15 dependent cell proliferation of 32D beta cells. The 18t15-12s complex exhibits reduced activity compared to human recombinant IL-15, probably due to the linkage of IL-18 and tissue factor to the IL-15 domain.

To assess the individual activities of IL-12 and IL-18 in the 18t15-12s complex, 18t15-12s was added to HEK-Blue IL-12 and HEK-Blue IL-18 reporter cells in 200. Mu.LIMDM: 10% heat-inactivated FBS medium (5X 10 ⁴ Individual cells/wells; hkb-il12 and hkb-hmil18, invivoGen). Cells were incubated overnight at 37 ℃. Mu.l of the induced HEK-Blue IL-12 and HEK-Blue IL-18 reporter cell supernatants were added to 180. Mu.l of QUANTI-Blue (InvivoGen) and incubated for 1-3 hours at 37 ℃. IL-12 or IL-18 activity was assessed by measuring absorbance at 620 nm. Human recombinant IL-12 or IL-18 was evaluated as positive or negative controls. As shown in fig. 30 and 31, each cytokine domain in the 18t15-12s complex retains a specific biological activity. The reduced activity of 18t15-12s compared to human recombinant IL-18 or IL-12 may be due to the linkage of IL-15 and tissue factor to the IL-18 domain and IL-12 to the IL-15Rα sushi domain.

Example 14: cytokine-induced memory-like NK cell production by the 18t15-12s complex

Cytokine-induced memory-like NK cells can be induced ex vivo after overnight stimulation of purified NK cells with saturated amounts of IL-12 (10 ng/mL), IL-15 (50 ng/mL) and IL-18 (50 ng/mL). These memory-like properties have been measured by expression of IL-2 receptor alpha (IL-2R alpha, CD 25), CD69 (and other activation markers) and increased IFN-gamma production. To assess the ability of the 18t15-12s complex to promote cytokine-induced memory-like NK cell production, purified human NK cells (> 95% CD56+) were stimulated with 0.01nM to 10000nM of 18t15-12s complex or a combination of individual cytokines (recombinant IL-12 (10 ng/mL), IL-18 (50 ng/mL) and IL-15 (50 ng/mL)) for 14-18 hours. Cell surface CD25 and CD69 expression and intracellular IFN- γ levels were assessed by antibody staining and flow cytometry.

Fresh human leukocytes were obtained from blood banks and cd56+ NK cells were isolated with rosetteep/human NK cell reagent (stem cell technology (StemCell Technologies)). Purity of NK cells>70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (hundred-Biotech). The cells were counted and at 0.2x10 ⁶ the/mL was resuspended in 0.2mL of complete medium (RPMI 1640 (Ji Bike (Gibco)) supplemented with 2mM L-glutamine (Semer Life technologies (Thermo Life Technologies)), penicillin (Semer Life technologies), streptomycin (Semer Life technologies) and 10% FBS (sea clone)) in 96 well flat bottom plates. At 37 ℃, 5% CO ₂ The cells were stimulated with a mixture of cytokines hIL-12 (10 ng/mL) (hundred-in Biotechnology), hIL-18 (50 ng/mL) (Andi organism) and hIL-15 (50 ng/mL) (NCI) or with 0.01nM to 10000nM 18t15-12s for 14-18 hours. Cells were then harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibody (hundred-Biotech) for 30 minutes. After staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD Millipore) and 0.001% sodium azide (sigma)) at 1500RPM for 5 min at room temperature. After washing twice, BD FACSCelesta was used ^TM Flow cytometry analyzed cells (plotted data-average fluorescence intensity; fig. 32A and fig. 32B).

Fresh human white from blood bankCells, and with Rosetteep/human NK cell reagent (Stem cell technology) separation of CD56+ NK cells. Purity of NK cells >70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (hundred-Biotech). The cells were counted and at 0.2x10 ⁶ the/mL was resuspended in 0.2mL of complete medium (RPMI 1640 (Ji Bike (Gibco)) supplemented with 2mM L-glutamine (Sesammer life technology), penicillin (Sesammer life technology), streptomycin (Sesammer life technology) and 10% FBS (sea clone)) in 96 well flat bottom plates. At 37℃at 5% CO ₂ hIL-12 (10 ng/mL) (hundred-in Biotechnology), hIL-18 (50 ng/mL) (Andi organism (R)&D) Cytokine mixture with hIL-15 (50 ng/mL) (NCI) or 18t15-12s complex of 0.01nM to 10000nM for 14-18 hours. Cells were then treated with 10. Mu.g/mL of Brefeldin A (sigma) and 1 Xmonensin (Monensin) (electronic bioscience) for 4 hours, then collected and stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies for 30 minutes. After staining, cells were washed in FACS buffer (1 XPBS (sea cloning), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature and fixed for 10 minutes at room temperature. After fixation, cells were washed in 1x permeabilization buffer (e-bioscience) for 5 min at 1500RPM at room temperature and stained with IFN- γ -PE Ab (hundred-bioscience) for 30 min at room temperature. The cells were again washed with 1x permeabilization buffer, then with FACS buffer. Cell pellet (Cell pellet) was resuspended in 300 μl FACS buffer and BD FACSCelesta was used ^TM Flow cytometry was used for analysis (percentage of IFN-. Gamma.positive cells plotted; FIG. 33).

Example 15: cytotoxicity of NK cells on human tumor cells in vitro

Human myelogenous leukemia cells K562 (CellTrace violet marker) were incubated with purified human NK cells in the presence of increasing concentrations of the 18t15-12s complex or cytokine mixture as control. After 20 hours, cultures were harvested, stained with Propidium Iodide (PI), and evaluated by flow cytometry. As shown in FIG. 34, the 18t15-12s complex induced cytotoxicity of K562 by human NK at a similar or greater level than the cytokine mixture, where both the 18t15-12s complex and the cytokine mixture induced greater cytotoxicity than the medium control.

Example 16: production of IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 DNA constructs

In one non-limiting example, IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 DNA constructs were generated (FIGS. 35 and 36). Human IL-12 subunit sequences, human IL-15RαSu sequences, human IL-15 sequences, human tissue factor 219 sequences and human IL-18 sequences were synthesized by Jin Weizhi. A DNA construct was made, with the GS (3) linker linking IL-12 subunit β (p 40) to IL-12 subunit α (p 35) to generate a single-stranded form of IL-12, directly linking the IL-12 sequence to the IL-15RαSu sequence, and directly linking the IL-12/IL-15RαSu construct to the N-terminal coding region of αCD16scFv.

The nucleic acid sequence of the IL-12/IL-15RαSu/αCD16scFv construct is as follows (SEQ ID NO: 226):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

(human IL-12 subunit beta (p 40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC

(Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human IL-12 subunit alpha (p 35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(anti-human CD16 light chain variable Domain)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT

(Joint)

GGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCC

(anti-human CD16 heavy chain variable domain)

GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

Constructs were also made as follows: the IL-18 sequence was ligated to the N-terminal coding region of tissue factor 219 and the IL-18/TF construct was ligated to the N-terminal coding region of IL-15 (FIG. 36). The nucleic acid sequence (including the leader sequence) of the IL-18/TF/IL-15 construct is as follows (SEQ ID NO: 177):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC

(human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCCExample 17: IL-12/IL-15RαSu/α Secretion of CD16scFv and IL-18/TF/IL-15 fusion proteins

IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 constructs were cloned into pMSGV-1 modified retroviral expression vectors (Hughes, hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vectors were then transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells resulted in secretion of the soluble IL-18/TF/IL-15:IL-12/IL-15RαSu/αCD16scFv protein complex (referred to as 18t15-12s/αCD16; FIGS. 37 and 38). Co-expression of both constructs in CHO-K1 cells resulted in secretion of soluble IL-18/TF/IL-15:IL-12/IL-15RαSu/αCD16scFv protein complexes (referred to as 18t15-12s/αCD16; FIGS. 37 and 38) which could be purified by anti-TF Ab affinity and other chromatographic methods. In some cases, the signal peptide is cleaved from the intact polypeptide to produce the mature form.

The amino acid sequence (including the signal peptide sequence) of the IL-12/IL-15RαSu/αCD16scFv fusion protein is as follows (SEQ ID NO: 225):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-12 subunit beta (p 40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

(Joint)

GGGGSGGGGSGGGGS

(human IL-12 subunit alpha (p 35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

(anti-human CD16 light chain variable Domain)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH

(Joint)

GGGGSGGGGSGGGGS

(anti-human CD16 heavy chain variable domain)

EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR

The amino acid sequence (including the leader sequence) of the IL-18/TF/IL-15 fusion protein is as follows (SEQ ID NO: 221):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 18: production of IL-18/IL-15RαSu and IL-12/TF/IL-15 DNA constructs

In a non-limiting example, IL-18/IL-15RαSu and IL-12/TF/IL-15 DNA constructs are produced. Human IL-18 subunit sequences, human IL-15RαSu sequences, human IL-12 sequences, human tissue factor 219 sequences and human IL-15 sequences were synthesized by Jin Weizhi. A DNA construct was made, connecting IL-18 directly to IL-15RαSu. Other constructs were also made, linking the IL-12 sequence to the N-terminal coding region in the form of human tissue factor 219, and further linking the IL-12/TF construct to the N-terminal coding region of IL-15. As described above, the single-stranded form of IL-12 (p 40-linker-p 35) was used.

The nucleic acid sequence (including the signal peptide sequence) of the IL-18/IL-15RαSu construct is shown below (SEQ ID NO: 320):

(Signal peptide)

ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC

(human IL-18)

TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The nucleic acid sequence (including the leader sequence) of the IL-12/TF/IL-15 construct is shown below (SEQ ID NO: 321):

(Signal peptide)

ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC

(human IL-12 subunit beta (p 40))

ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC

(Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human IL-12 subunit alpha (p 35))

CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Example 19: secretion of IL-18/IL-15RαSu and IL-12/TF/IL-15 fusion proteins

IL-18/IL-15RαSu and IL-12/TF/IL-15 constructs were cloned into pMSGV-1 modified retroviral expression vectors (Hughes, hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vectors transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells resulted in secretion of the soluble IL-12/TF/IL-15:IL-18/IL-15RαSu protein complex (called 12t15/s 18) which could be purified by anti-TF Ab affinity and other chromatographic methods.

The amino acid sequence (including the signal peptide sequence) of the IL-18/IL-15RαSu fusion protein is shown below (SEQ ID NO: 322):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-18)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence (including the leader sequence) of the IL-12/TF/IL-15 fusion protein is shown below (SEQ ID NO: 323):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-12 subunit beta (p 40))

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS

(Joint)

GGGGSGGGGSGGGGS

(human IL-12 subunit alpha (p 35))

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

Example 20: quantification of recombinant proteins of the 18t15-12s16 Complex

The 18t15-12s16 complex (comprising IL-12/IL-15RαSu/αCD16scFv; IL-18/TF/IL-15) was detected and quantified using standard sandwich ELISA methods (FIG. 39). Anti-human tissue factor antibody/IL-2 or anti-TF Ab/IL-18 served as capture antibodies, and biotinylated anti-human IL-12 or IL-18 antibodies (BAF 219, D045-6, all from Andi organisms) served as detection antibodies. Tissue factor is also detected using an anti-human tissue factor antibody (I43) and an anti-human tissue factor antibody detection antibody.

Example 21: production of TGF-beta RII/IL-15RαSu and IL-21/TF/IL-15 DNA constructs

In one non-limiting example, a TGF-beta RII/IL-15R alpha Su DNA construct is generated (FIG. 40). Human TGF-beta RII dimer and human IL-21 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Preparing a DNA construct, ligating tgfbetarii to another tgfbetarii with a linker to produce a single-stranded form of tgfbetarii, and then ligating the tgfbetarii single-stranded dimer sequence directly to the N-terminal coding region of IL-15rαsu.

The nucleic acid sequence (including the signal sequence) of the TGF-beta RII/IL-15RαSu construct is as follows (SEQ ID NO: 196):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF beta RII-fragment 1)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT

(Joint)

GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT

(human TGF beta RII-fragment 2)

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human IL-15Rα sushi Domain)

ATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA

In addition, IL-21/TF/IL-15 constructs were made as follows: the IL-21 sequence was linked to the N-terminal coding region of tissue factor 219, and the IL-21/TF construct was further linked to the N-terminal coding region of IL-15 (FIG. 41). The nucleic acid sequence (including the leader sequence) of the IL-21/TF/IL-15 construct is as follows (SEQ ID NO: 192):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human tissue factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Example 22: secretion of TGF-beta RII/IL-15RαSu and IL-21/TF/IL-15 fusion proteins

TGFβRII/IL-15RαSu and IL-21/TF/IL-15 DNA constructs were cloned into pMSGV-1 modified retroviral expression vectors (as described in Hughes, hum Gene Ther16:457-72, 2005, incorporated herein by reference) and the expression vectors transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells resulted in secretion of the soluble IL-21/TF/IL-15 TGF-beta RII/IL-15R alpha Su protein complex (referred to as 21t15-TGFRs; FIGS. 42 and 43). The 21t15-TGFRs complex was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and other chromatographic methods.

The amino acid sequence (including the signal peptide sequence) of the TGF-beta RII/IL-15RαSu construct is as follows (SEQ ID NO: 195):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF beta RII-fragment 1)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(Joint)

GGGGSGGGGSGGGGS

(human TGF beta RII-fragment 2)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence (including the signal peptide sequence) of the mature IL-21/TF/IL-15 fusion protein is as follows (SEQ ID NO: 191):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

Example 23: purification of 21t15-TGFRs by immunoaffinity chromatography

Attachment of anti-TF antibody affinity columns to general electric medical community AKTA ^TM Avant protein purification system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Cell culture collections of 21t15-TGFRs were adjusted to pH 7.4 with 1M Tris base and loaded onto anti-TF antibody affinity columns equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). Absorbance at 280nm was collected and then the sample was then neutralized to pH 7.5-8.0 by adding 1MTris base. Then using a molecular weight cut-off value of 30kDa The filter was centrifuged and the neutralized sample was buffer exchanged in PBS. FIG. 44 shows that the 21t15-TGFRs complex binds to an anti-TF antibody affinity column where TF is 21t15-TGFRs binding partners. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 10 column volumes of PBS, 0.05% sodium azide, and stored at 2-8 ℃.

Example 24: size exclusion chromatography of 21t15-TGFRs

General electric medical group200 The Increase 10/300 GL gel filtration column was connected to the general electric medical community AKTA ^TM Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8mL/min. 200. Mu.L of 1mg/mL 21t15-TGFRs complex was injected onto the column using a capillary loop. The injections were then followed by 1.25 column volumes of PBS. SEC chromatography is shown in figure 45. There are two protein peaks, probably representing monomeric and dimeric forms of 21t 15-TGFRs.

Example 25: SDS-PAGE of 21t15-TGFRs

To determine purity and protein molecular weight, purified 21t15-TGFRs complex protein samples were analyzed under reducing conditions using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel was treated with InstantBuue ^TM Dyeing for about 30min, and decolorizing in purified water overnight. FIG. 46 shows an exemplary SDS gel of affinity purified 21t15-TGFRs of anti-TF antibodies with bands at 39.08kDa and 53 kDa.

Glycosylation of 21t15-TGFRs in CHO cells was confirmed using protein deglycosylation mixture II kit (New England Biolabs) and manufacturer's instructions. Deglycosylation reduces the molecular weight of 21t15-TGFRs as shown in fig. 46 lane 4.

Example 26: quantification of recombinant proteins of the 21t15-TGFRs Complex

The 21t15-TGFRs complex was detected and quantified using standard sandwich ELISA methods (FIGS. 47-50). The anti-human tissue factor antibody serves as a capture antibody and the biotinylated anti-human IL-21, IL-15 or TGF-beta RII serves as a detection antibody. Tissue factor is also detected using an anti-human tissue factor capture antibody (I43) and an anti-human tissue factor antibody detection antibody. The I43/anti-TF antibody ELISA was compared to purified tissue factor at similar concentrations.

Example 27: immunostimulatory capacity of the 21t15-TGFRs Complex

To evaluate IL-15 immunostimulatory activity of 21t15-TGFRs complexes, 32D beta cells (10 ⁴ Individual cells/well) was added with increasing concentrations of 21t15-TGFRs and the cells were incubated at 37 ℃ for 3 days. On the fourth day, WST-1 proliferation reagent (10. Mu.L/well) was then added, and after 4 hours, absorbance was measured at 450nm to determine cell proliferation based on cleavage of WST-1 into soluble formazan dye. The biological activity of human recombinant IL-15 was evaluated as a positive control. As shown in FIG. 51, 21t15-TGFRs exhibit IL-15 dependent 32D beta cell proliferation. The activity of the 21t15-TGFRs complex is reduced compared to human recombinant IL-15, probably due to the linkage of IL-21 and tissue factor to the IL-15 domain.

In addition, HEK-Blue TGF-beta reporter cells (hkb-tgfb, invivoGen) were used to measure the ability of 21t15-TGFRs to block TGF-beta 1 activity (FIG. 52). 21t15-TGFRs at increasing concentrations were mixed with 0.1nM TGF-beta 1 and added to 200. Mu.L of HEK-Blue TGF-beta reporter cells (2.5X10 s) in 10% heat-inactivated FBS medium ⁴ Individual cells/well). Cells were incubated overnight at 37 ℃. The next day, 20. Mu.l of induced HEK-Blue TGF-beta reporter cell supernatant was added to 180. Mu.l of QUANTI-Blue ((InvivoGen) and incubated at 37℃for 1-3 hours.

These results indicate that the tgfbetarii domain of the 21t15-TGFRs complex retains its ability to capture tgfbeta 1. The reduced ability of 21t15-TGFRs to block TGF-beta 1 activity compared to human recombinant TGF-beta RII/Fc is likely due to the ligation of TGF-beta RII to the IL-15Rα sushi domain.

Examples28: cytokine induced memory-like NK cell production by 21t15-TGFRs complex

Cytokine-induced memory-like NK cells can be induced ex vivo after overnight stimulation of purified NK cells with a saturated amount of cytokine. These memory-like properties can be measured by expression of IL-2 receptor alpha (IL-2R alpha, CD 25), CD69 (and other activation markers), and increased IFN-gamma production. To assess the ability of the 21t15-TGFRs complex to promote cytokine-induced memory-like NK cell production, purified human NK cells (> 95% cd56+) were stimulated with 1nM to 100nM of the 21t15-TGFRs complex for 14-18 hours. Cell surface CD25 and CD69 expression and intracellular IFN- γ levels were assessed by antibody staining and flow cytometry.

Fresh human leukocytes were obtained from blood banks and cd56+ NK cells were isolated with rosetteep/human NK cell reagent (stem cell technology). Purity of NK cells>70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (hundred-Biotech). The cells were counted and at 0.2x10 ⁶ the/mL was resuspended in 0.2mL complete medium (RPMI 1640 (Ji Bike) supplemented with 2mM L-glutamine (Sesammer life technologies), penicillin (Sesammer life technologies), streptomycin (Sesammer life technologies) and 10% FBS (sea clones)) in 96 well flat bottom plates. Cells were stimulated overnight with a mixed cytokine of hIL-21 (50 ng/ml) (hundred-in Biotechnology) and hIL-15 (50 ng/ml) (NCI) or with 1nM, 10nM or 100nM of 21t15-TGFRs complex at 37℃for 14-18 hours. Cells were then harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies for 30 minutes. After staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After washing twice, BD FACSCelesta was used ^TM Flow cytometry analyzes cells. (plotted data-average fluorescence intensity; fig. 53 and 54).

Fresh human leukocytes were obtained from blood banks and cd56+ NK cells were isolated using rosetteep/human NK cell reagent (stem cell technology). Purity of NK cells>70, andand confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (hundred-Biotech). Cells were counted and resuspended at 0.2×106/mL in 0.2mL of complete medium (RPMI 1640 (Ji Bike), supplemented with 2mM L-glutamine (sammer life technology), penicillin (sammer life technology)), streptomycin (sammer life technology) and 10% fbs (sea clones)) in flat bottom 96-well plates. Cells were stimulated overnight with either a mixed cytokine of hIL-21 (50 ng/ml) (hundred-in Biotechnology) and hIL-15 (50 ng/ml) (NCI) or 1nM, 10nM or 100nM of the 21t15-TGFRs complex at 37℃for 14-18 hours. Cells were then treated with 10. Mu.g/ml of brefeldin A (sigma) and 1 Xmonensin (electronic bioscience) for 4 hours. Cells were harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies for 30 minutes. After staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature and fixed for 10 minutes at room temperature. After fixation, cells were washed with 1x permeabilization buffer (e-bioscience) (1500 RPM at room temperature for 5 min) and stained with intracellular IFN- γ -PE (hundred-bioscience) for 30 min at room temperature. The cells were again washed with 1x permeabilization buffer, then with FACS buffer. The cell pellet was resuspended in 300 μl FACS buffer and BD FACSCelesta was used ^TM Flow cytometry was used for analysis. (percentage of IFN-. Gamma.positive cells plotted; FIG. 55).

Example 29: cytotoxicity of NK cells on tumor cells in vitro

Human myelogenous leukemia cells K562 (CellTrace violet tag) were incubated with purified human NK cells in the presence of increasing concentrations of 21T15-TGFRs complex (using the StemCell human NK cell purification kit (E: T ratio; 2: 1)). After 20 hours, cultures were harvested, stained with Propidium Iodide (PI), and evaluated by flow cytometry. As shown in fig. 56, the 21t15-TGFRs complex induces cytotoxicity of human NK against K562 as compared to the control.

Examples30: production of IL-21/TF mutant/IL-15 DNA constructs and TGF-beta RII/IL-15RαSu TGF-derived fusion protein complexes

In a non-limiting example, an IL-21/TF mutant/IL-15 DNA construct is prepared by ligating IL-21 directly to the N-terminal coding region of a tissue factor 219 mutant, and further ligating an IL-21/TF mutant to the N-terminal coding region of IL-15.

The nucleic acid sequence (including the signal peptide sequence) of the IL-21/TF mutant/IL-15 construct is as follows (SEQ ID NO:324, shaded nucleotides are mutants, and mutation codons are underlined):

(Signal sequence)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human tissue factor 219 mutant)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCGCGACAGCTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGCTTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGGCGCGGAATAACACAGCTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the signal peptide sequence) of the IL-21/TF mutant/IL-15 construct is as follows (SEQ ID NO:325, substituted residues shaded):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

In some embodiments, the IL-21/TF mutant/IL-15 DNA construct can be combined with a TGF-beta RII/IL-15RαSu DNA construct, transfected into cells using a retroviral vector as described above, and expressed as an IL-21/TF mutant/IL-15 and TGF-beta RII/IL-15RαSu fusion protein. The IL-15RαSu domain of the TGFβRII/IL-15RαSu fusion protein binds to the IL-15 domain of the IL-21/TF mutant/IL-15 fusion protein to produce an IL-21/TF mutant/IL-15:TGFβRII/IL-15RαSu complex.

Example 31: production of IL-21/IL-15RαSu and TGFβRII/TF/IL-15DNA constructs and resulting fusion proteins White composite

In a non-limiting example, the IL-21/IL-15RαSu DNA construct is prepared by directly ligating IL-21 to the IL-15RαSu subunit sequence. The nucleic acid sequence (including the signal sequence) of the IL-21/IL-15RαSu construct is as follows (SEQ ID NO: 214):

(Signal sequence)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence (including the signal peptide sequence) of the IL-21/IL-15RαSu construct is as follows (SEQ ID NO: 213):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

In some embodiments, the IL-21/IL-15RαSu DNA construct may be combined with a TGF- βRII/TF/IL-15 DNA construct, transfected into a retroviral vector as described above, and expressed as IL-21/IL-15RαSu and TGF- βRII/TF/IL-15 fusion proteins. The IL-15RαSu domain of the IL-21/IL-15RαSu fusion protein binds to the IL-15 domain of the TGF-beta RII/TF/IL-15 fusion protein to produce a TGF-beta RII/TF/IL-15:IL-21/IL-15RαSu complex.

TGF-beta RII/TF/IL-15R alpha Su DNA constructs are generated by ligating the TGF-beta RII sequence to the N-terminal coding region of human tissue factor 219 form, and then ligating the TGF-beta RII/TF construct to the N-terminal coding region of IL-15. As described above, a single-chain form of TGF-beta RII (TGF-beta RII-linker-TGF-beta RII) is used. The nucleic acid sequence (including the leader sequence) of the TGF-beta RII/TF/IL-15 construct is as follows (SEQ ID NO: 239):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF beta RII-fragment 1)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT

(Joint)

GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT

(human TGF beta RII-fragment 2)

ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence of the TGF-beta RII/TF/IL-15 fusion protein (including the signal peptide) is as follows (SEQ ID NO: 238):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF beta RII-fragment 1)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(Joint)

GGGGSGGGGSGGGGS

(human TGF beta RII-fragment 2)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 32 production of exemplary Single-chain chimeric Polypeptides

An exemplary single chain chimeric polypeptide was generated that included a first target binding domain that was an anti-CD 3scFv, a soluble human tissue factor domain, and a second target binding domain that was an anti-CD 28scFv (αcd3scfv/TF/αcd28scfv) (fig. 57). The nucleic acid and amino acid sequences of such single-chain chimeric polypeptides are shown below.

Nucleic acid encoding an exemplary single chain chimeric polypeptide (aCD 3 scFv/TF/aCD 28 scFv) (SEQ ID NO: 158)

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCC

(αCD3 light chain variable region)

CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGT

(Joint)

GGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGC

(αCD3 heavy chain variable region)

CAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACTATAACCAAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC

(human tissue factor 219 form)

TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(αCD28 light chain variable region)

GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC

(Joint)

GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCC

(αCD28 light chain variable region)

GACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG

Exemplary Single chain chimeric polypeptide (αCD3scFv/TF/αCD28 scFv) (SEQ ID NO: 157)

(Signal peptide)

MKWVTFISLLFLFSSAYS

(αCD3 light chain variable region)

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR

(Joint)

GGGGSGGGGSGGGGS

(αCD3 heavy chain variable region)

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(αCD28 light chain variable region)

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSS

(Joint)

GGGGSGGGGSGGGGS

(αCD28 heavy chain variable region)

DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR

A second exemplary single chain chimeric polypeptide was generated that included a first target binding domain that was an anti-CD 28scFv, a soluble human tissue factor domain, and a second target binding domain that was an anti-CD 3scFv (αcd28scFv/TF/αcd3 scFv) (fig. 57). The nucleic acid and amino acid sequences of such single-chain chimeric polypeptides are shown below.

Nucleic acid (SEQ ID NO: 326) encoding an exemplary single chain chimeric polypeptide (αCD28scFv/TF/αCD3 scFv)

(Signal peptide)

ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCTACAGC

(αCD28 light chain variable region)

GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCC

(Joint)

GGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGC

(αCD28 heavy chain variable region)

GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGG

(human tissue factor 219 form)

AGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAG

(αCD3 light chain variable region)

CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGT

(Joint)

GGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGC

(αCD3 heavy chain variable region)

CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC

Exemplary Single chain chimeric polypeptide (αCD28scFv/TF/αCD3 scFv) (SEQ ID NO: 327)

(Signal peptide)

MKWVTFISLLFLFSSAYS

(αCD28 light chain variable region)

VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSS

(Joint)

GGGGSGGGGSGGGGS

(αCD28 heavy chain variable region)

DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(αCD3 light chain variable region)

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR

(Joint)

GGGGSGGGGSGGGGS

(αCD3 heavy chain variable region)

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

Nucleic acids encoding the αCD3scFv/TF/αCD28scFv were cloned into modified retroviral expression vectors as previously described (Hughes et al, hum Gene Ther 16:457-72, 2005). Expression vectors encoding αCD3scFv/TF/αCD28scFv were transfected into CHO-K1 cells. Expression of the expression vector in CHO-K1 cells allows secretion of a soluble αcd3scfv/TF/αcd28scfv single chain chimeric polypeptide (designated 3t 28), which can be purified by anti-TF Ab affinity and other chromatographic methods.

An anti-tissue factor affinity column was used to purify the αcd3scFv/TF/αcd28scFv single chain chimeric polypeptide. The anti-tissue factor affinity column was connected to the universal electric medical community AKTA Avant system. All steps except the elution step used a flow rate of 4mL/min, with the flow rate of the elution step being 2mL/min.

Cell culture collections containing αcd3scFv/TF/αcd28scFv single chain chimeric polypeptides were adjusted to pH 7.4 using 1M Tris base and loaded onto an anti-TF antibody affinity column (described above) equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 by addition of 1M Tris base. The neutralized sample buffer was then exchanged into PBS using an Amicon centrifuge filter with a molecular weight cut-off of 30 kDa. The data in fig. 58 shows that the anti-tissue factor affinity column is capable of binding to an αcd3scFv/TF/αcd28scFv single chain chimeric polypeptide comprising a human soluble tissue factor domain. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing.

After each elution, the anti-tissue factor affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). Then 10 column volumes of PBS, 0.05% NaN were used ₃ The column was neutralized and stored at 2-8 ℃.

Analytical Size Exclusion Chromatography (SEC) was performed on αcd3scFv/TF/αcd28scFv single chain chimeric polypeptides using a Superdex200 increment 10/300 GL gel filtration column (from the general electric healthcare group) attached to the AKTA Avant system (from the general electric healthcare group). The column was equilibrated with 2 column volumes of PBS. A flow rate of 0.8mL/min was used. Two hundred microliters (μl) of the αcd3scfv/TF/αcd28scfv single chain chimeric polypeptide (1 mg/mL) was injected onto the column using a capillary loop. After injection of the single-stranded chimeric polypeptide, 1.25 column volumes of PBS were flowed into the column. SEC chromatograms are shown in figure 59. The data shows that there are 3 protein peaks, possibly representing monomers and dimers or other different forms of the αcd3scFv/TF/αcd28scFv single chain chimeric polypeptide.

To determine the purity and protein molecular weight of the αcd3scFv/TF/αcd28scFv single chain chimeric polypeptides, αcd3scFv/TF/αcd28scFv protein samples purified from anti-tissue factor affinity columns were analyzed under reducing conditions by standard sodium dodecyl sulfate polyacrylamide gel (4-12% nupage Bis-Tris gel) electrophoresis (SDS-PAGE) method. The gel was stained with InstantBuue for about 30 minutes and destained with purified water overnight. FIG. 60 shows SDS gels of single-chain chimeric polypeptides of αCD3scFv/TF/αCD28scFv purified using an anti-tissue factor affinity column. The results indicate that the purified αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide has the expected molecular weight (72 kDa) in reduced SDS gels.

Example 33.αFunctional characterization of CD3scFv/TF/αCD28scFv single-chain chimeric polypeptides

ELISA-based methods confirmed the formation of the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptides. The αcd3scfv/TF/αcd28scfv single chain chimeric polypeptide was detected using a capture antibody, an anti-human tissue factor antibody (I43), and a detection antibody, anti-TF antibody, using an anti-TF antibody (I43)/anti-TF antibody specific ELISA (fig. 61). Purified tissue factor protein at similar concentrations was used as a control.

Further in vitro experiments were performed to determine if the αcd3scFv/TF/αcd28scFv single chain chimeric polypeptide was capable of activating human Peripheral Blood Mononuclear Cells (PBMCs). Fresh human leukocytes were obtained from a blood pool and Peripheral Blood Mononuclear Cells (PBMCs) were isolated using a density gradient Histopaque (sigma). Cells were counted and at 0.2x10 ⁶ A concentration of/mL was resuspended in 0.2mL of complete medium (RPMI 1640 (Ji Bike) supplemented with 2mM L-glutamine (Sesammer life technology), penicillin (Sesammer life technology)) in flat bottom 96 well plates,Streptomycin (sammer life technology) and 10% bs (sea clone)). At 37℃at 5% CO ₂ Cells were stimulated with 0.01nM to 1000nM of the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide for 3 days. After 72 hours, cells were harvested and surface stained with CD4-488, CD8-PerCP Cy5.5, CD25-BV421, CD69-APCFire750, CD62L-PE Cy7 and CD44-PE specific antibodies (hundred-in Biotechnology) for 30 minutes. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After two washes, the cells were resuspended in Is performed by flow cytometry (Celesta-BD Bioscience). The data in FIGS. 62 and 63 show that the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide is capable of stimulating CD8 ⁺ And CD4 ⁺ T cells.

Further experiments were performed in which PBMC isolated using Histopaque (Sigma) were counted and at 0.2x10 ⁶ the/mL was resuspended in 0.2mL of complete medium (RPMI 1640 (Ji Bike) supplemented with 2mM L-glutamine (Sesammer life technologies), penicillin (Sesammer life technologies), streptomycin (Sesammer life technologies) and 10% FBS (sea clones)) in flat bottom 96-well plates. Then at 37℃at 5% CO ₂ Cells were stimulated with 0.01nM to 1000nM of the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide for 3 days. After 72 hours, cells were harvested and the surface was stained with CD4-488, CD8-PerCP Cy5.5, CD25-BV421, CD69-APCFire750, CD62L-PE Cy7 and CD44-PE (hundred-in Biotechnology) for 30 minutes. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After two washes, the cells were resuspended inIs performed by flow cytometry (Celesta-BD biosciences). The data again indicate that the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide is capable of stimulating CD4 ⁺ T cellActivation (fig. 64).

Example 34: IL-7/IL-15RαSu Production of DNA constructs

In one non-limiting example, an IL-7/IL-15RαSu DNA construct is generated (see FIG. 65). Human IL-7 sequences, human IL-15RαSu sequences, human IL-15 sequences and human tissue factor 219 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. DNA constructs were made, linking the IL-7 sequence to the IL-15RαSu sequence. The final IL-7/IL-15RαSu DNA construct sequence was synthesized by Jin Weizhi.

The nucleic acid sequence (including the signal peptide sequence) encoding the second chimeric polypeptide of the IL-7/IL-15RαSu construct is shown below (SEQ ID NO: 206):

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC

(human IL-7)

GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC

(human IL-15Rα sushi Domain)

ATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGA

The second chimeric polypeptide (including the signal peptide sequence) of the IL-7/IL-15RαSu construct is shown below (SEQ ID NO: 205):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 35: production of IL-21/TF/IL-15 DNA constructs

In one non-limiting example, an IL-21/TF/IL-15 construct is made by ligating an IL-21 sequence to the N-terminal coding region of tissue factor 219, and further ligating the IL-21/TF construct to the N-terminal coding region of IL-15 (FIG. 66).

The nucleic acid sequence (including the leader sequence) encoding the first chimeric polypeptide of the IL-21/TF/IL-15 construct synthesized by Jin Weizhi is shown below (SEQ ID NO: 202):

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC

(human IL-21 fragment)

CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATGTATCAATTAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCC

(human tissue factor 219)

TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide comprising the IL-21/TF/IL-15 construct of the leader sequence is SEQ ID NO. 201:

(Signal peptide)

MGVKVLFALICIAVAEA(SEQ ID NO:328)

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 36: secretion of IL-7/IL-15RαSu and IL-21/TF/IL-15 fusion proteins

IL-7/IL-15RαSu and IL-21/TF/IL-15 DNA constructs were cloned into pMSGV-1 modified retroviral expression vectors (Hughes, hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vectors transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allowed the formation and secretion of soluble IL-21/TF/IL-15:IL-7/IL-15RαSu protein complexes (referred to as 21t15-7s; FIGS. 67 and 68). The 21t15-7s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody affinity chromatography and size exclusion chromatography to give a soluble (non-aggregate) protein complex consisting of IL-7/IL-15rαsu and IL-21/TF/IL-15 fusion proteins.

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

Example 37: purification of 21t15-7s by immunoaffinity chromatography

Attaching anti-TF antibody affinity columns to general electric medical group (GE Healthcare) ^TM ) AKTA Avant protein purification system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Cell culture collections from 21t15-7s were adjusted to pH 7.4 with 1M Tris base and loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). Absorbance at 280nm was collected and the samples were then neutralized to pH 7.5-8.0 by adding 1M Tris base. Then using a molecular weight cut-off value of 30kDaThe centrifuge filter exchanges the neutralized sample buffer into PBS. Buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was then stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 10 column volumes of PBS, 0.05% sodium azide, and stored at 2-8 ℃.

Example 38: size exclusion chromatography

General electric medical group200 The Increase 10/300 GL gel filtration column was connected to the general electric medical community AKTA ^TM Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7mL/min. 200. Mu.L of 1mg/mL of the 7t15-21s complex was injected onto the column using a capillary loop. The injections were followed by 1.25 column volumes of PBS.

Example 39: SDS-PAGE of 21t15-7s and 21t15-TGFRs

To determine purity and protein molecular weight, purified 21t15-7s or 21t15-TGFRs protein samples were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE. The gel will be treated with InstantBuue ^TM Staining for about 30min followed by decolorization in purified water overnight.

Example 40: 21t15-7s and 21t15-TGFRs glycosylation in CHO-K1 cells

21t15-7s glycosylation in CHO-K1 cells or 21t15-TGFRs glycosylation in CHO-K1 cells was confirmed using protein deglycosylation mixture II kit (New England Biolabs) according to manufacturer's instructions.

Example 41: quantification of recombinant proteins of the 21t15-7s and 21t15-TGFRs complexes

The 21t15-7s complex or 21t15-TGFRs complex was detected and quantified using standard sandwich ELISA methods. An anti-human tissue factor antibody (IgG 1) serves as a capture antibody and a biotinylated anti-human IL-21, IL-15 or IL-7 antibody (21 t15-7 s) or a biotinylated anti-human IL-21, IL-15 or TGF-beta RII antibody (21 t 15-TGFRs) serves as a detection antibody. The tissue factor in the purified 21t15-7s or 21t15-TGFRs protein complex was detected using an anti-human tissue factor capture antibody and an anti-human tissue factor antibody (IgG 1) detection antibody. The anti-TF antibody ELISA was compared to purified tissue factor at similar concentrations.

Example 42: production of IL-21/IL-15RαSu DNA constructs

In a non-limiting example, an IL-21/IL-15RαSu DNA construct is generated. The human IL-21 sequence and the human IL-15RαSu sequence were obtained from the UniProt website, and the DNA of these sequences was synthesized by Jin Weizhi. A DNA construct was made, linking the IL-21 sequence to the IL-15RαSu sequence. The final IL-21/IL-15RαSu DNA construct sequence was synthesized by Jin Weizhi. See fig. 69.

Example 43: production of IL-7/TF/IL-15 DNA constructs

In one non-limiting embodiment, the IL-7/TF/IL-15 construct is made by ligating the IL-7 sequence to the N-terminal coding region of tissue factor 219 and further ligating the IL-7/TF construct to the N-terminal coding region of IL-15. See fig. 70.

Example 44: production of IL-21/IL-15Rα Sushi domain DNA constructs

In a non-limiting example, a second chimeric polypeptide of IL-21/IL-15RαSu is produced. Human IL-21 and human IL-15Rα sushi sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. A DNA construct was made, linking the IL-21 sequence to the IL-15Rα sushi sequence. The final IL-21/IL-15RαSu DNA construct sequence was synthesized by Jin Weizhi.

The nucleic acid sequence (including the leader sequence) of the second chimeric polypeptide encoding the IL-21/IL-15RαSu domain synthesized by Jin Weizhi is shown below (SEQ ID NO: 214):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The second chimeric polypeptide (including the leader sequence) of the IL-21/IL-15Rα sushi domain is shown below (SEQ ID NO: 213):

(Signal sequence)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

Example 45: production of IL-7/TF/IL-15 DNA constructs

In a non-limiting example, an exemplary first chimeric polypeptide of IL-7/TF/IL-15 is prepared by ligating an IL-7 sequence to the N-terminal coding region of tissue factor 219, and further ligating an IL-7/TF construct to the N-terminal coding region of IL-15. The nucleic acid sequence (including the leader sequence) encoding the first chimeric polypeptide of IL-7/TF/IL-15 synthesized by Jin Weizhi is shown below (SEQ ID NO: 210):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-7 fragment)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The first chimeric polypeptide of IL-7/TF/IL-15 (comprising the leader sequence) is shown below (SEQ ID NO: 209):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 46: secretion of IL-21/IL-15RαSu and IL-7/TF/IL-15 fusion proteins

IL-21/IL-15RαSu and IL-7/TF/IL-15 DNA constructs were cloned into pMSGV-1 modified retroviral expression vectors (as described in Hughes, hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vectors transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells results in the formation and secretion of soluble IL-7/TF/IL-15:IL-21/IL-15RαSu protein complexes (referred to as 7t15-21 s). 7t15-21s protein was purified from CHO-K1 cell culture supernatant using anti-TF antibody (IgG 1) affinity chromatography and size exclusion chromatography to give a soluble (non-aggregate) protein complex consisting of IL-21/IL-15RαSu and IL-7/TF/IL-15 fusion proteins. See fig. 71 and 72.

Example 47: expansion ability of 7t15-21s Complex+anti-TF IgG1 antibodies to Primary Natural Killer (NK) cells

To assess the ability of the 7t15-21s complex to expand primary Natural Killer (NK) cells, the 7t15-21s complex and the 7t15-21s complex+anti-TF IgG1 antibodies were added to NK cells obtained from fresh human leukocyte samples. At 37℃and 5% CO ₂ Cells were stimulated with 50nM of the 7t15-21s complex with or without 25nM of anti-TF IgG1 or anti-TF IgG4 antibodies. Cells were maintained at 0.5x10 by counting every 48-72 hours ⁶ Per mL, no more than 2.0x10 ⁶ concentration/mL, and medium was supplemented with fresh stimulant. Cells stimulated with the 7t15-21s complex or anti-TF IgG1 antibody or anti-TF IgG4 antibody or anti-TF IgG4+7t15-21s complex were maintained until day 5. Primary NK cells were observed to expand after incubation with 21t15-7s complex + anti-TF IgG1 antibody.

Example 48: NK cells activated and amplified by 7t15-21s complex and anti-TF IgG1 antibody

After overnight stimulation of purified NK cells with 7t15-21s complex + anti-TF IgG1 antibody, primary NK cells were induced ex vivo. Obtained from blood banksFresh human leukocytes were obtained and CD56+ NK cells were isolated with Rosetteep/human NK cell reagent (Stem cell technology). Purity of NK cells >80% and confirmed by staining with antibodies specific for CD56-BV421 and CD16-BV510 (hundred-in biotechnology). Cells were counted and at 1x10 ⁶ 1mL of complete medium (RPMI 1640 (Ji Bike), supplemented with 4mM L-glutamine (Semer life technologies), penicillin (Semer life technologies), streptomycin (Semer life technologies), nonessential amino acids (Semer life technologies), sodium pyruvate (Semer life technologies) and 10% FBS (sea clones)) was resuspended in flat bottom 24-well plates. At 37℃and 5% CO ₂ Cells were stimulated with 25nM of anti-TF IgG1 antibody with or without 50nM of 7t15-21 s. Cells were counted every 48-72 hours and maintained at 0.5x10 ⁶ /mL to 2.0x10 ⁶ Concentration of/mL until day 14. The medium was periodically replenished with fresh stimulant. Cells were harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies on day 3 (hundred in biosciences and analyzed by flow cytometry-Celese-BD Bioscience). The activation marker CD25 MFI was observed to increase with 7t15-21s complex + anti-TF IgG1 antibody stimulation, but not with 7t15-21s complex stimulation. The activation marker CD69 MFI was observed to increase with the 7t15-21s complex + anti-TF IgG1 antibody and 7t15-21s complex alone.

Example 49: use of 18t15-12s to increase glucose metabolism in NK cells

A set of experiments was performed to determine the effect of the 18t15-12s construct on the oxygen consumption rate and extracellular acidification rate (ECAR) of NK cells purified from human blood.

In these experiments, fresh human leukocytes were obtained from two different human donors in a blood bank, and NK cells were isolated by negative selection using RosetteSep/human NK cell reagent (stem cell technology). Purity of NK cells>80% and confirmed by staining with antibodies specific for CD56-BV421 and CD16-BV510 (hundred-in biotechnology). The cells were counted and kept at 2x10 ⁶ 1mL of complete medium (RPMI 1640 (Ji Bike), supplemented withFilled with 4mM L-glutamine (Sesammer life technology), penicillin (Sesammer life technology), streptomycin (Sesammer life technology), nonessential amino acids (Sesammer life technology), sodium pyruvate (Sesammer life technology) and 10% FBS (sea clone)). At 37℃and 5% CO ₂ Cells were stimulated overnight with (1) medium alone, (2) 100nM 18t15-12s or (3) a mixture of single cytokine recombinant human IL-12 (0.25 μg), recombinant human IL-15 (1.25 μg) and recombinant human IL-18 (1.25 μg). The next day, cells were harvested and extracellular flux analysis was performed on the expanded NK cells using XFp analyzer (Seahorse Bioscience). The harvested cells were washed and washed at 2.0x10 ⁵ Individual cells/wells were plated at least in duplicate for extracellular flux analysis for OCR (oxygen consumption rate) and ECAR (extracellular acidification rate). Glycolytic stress testing was performed in Seahorse medium containing 2mM glutamine. The following substances were used in the analysis: 10mM glucose; 100nM oligomycin; and 100mM 2-deoxy-D-glucose (2 DG).

The data show that 18t15-12s resulted in a significant increase in oxygen consumption rate (FIG. 73) and extracellular acidification rate (ECAR) compared to the same cells activated with the combination of recombinant human IL-12, recombinant human IL-15 and recombinant human IL-18 (FIG. 74).

Example 50: generation and characterization of 7t15-16s21 fusion proteins

A fusion protein complex is generated comprising anti-CD 16scFv/IL-15rαsu/IL-21 and IL-7/TF/IL-15 fusion proteins. Human IL-7 and IL-21 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were made that ligate the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by ligation to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of constructs comprising IL-7 linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the IL-7/TF/IL-15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the IL-7/TF/IL-15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by ligating the human CD16scFv sequence to the N-terminal coding region of the IL-15RαSu chain, followed by ligating to the N-terminal coding region of IL-21 synthesized by Jin Weizhi. The nucleic acid and protein sequences of constructs comprising an anti-CD 16scFv linked to the N-terminus of the IL-15RαSu chain followed by the N-terminal coding region of IL-21 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the anti-CD 16SscFv/IL-15RαSu/IL-21 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

((anti-human CD16 scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence (including the signal peptide sequence) of the anti-CD 16scFv/IL-15RαSu/IL-21 construct is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(anti-human CD16 scFv)

seltqdpavsvalgqtvritcqgdslrsyyaswyqqkpgqapvlviygknnrpsgipdrfsgsssgntasltitgaqaedeadyycnsrdssgnhvvfgggtkltvghggggsggggsggggsevqlvesgggvvrpggslrlscaasgftfddygmswvrqapgkglewvsginwnggstgyadsvkgrftisrdnaknslylqmnslraedtavyycargrsllfdywgqgtlvtvsr

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

The anti-CD 16scFv/IL-15RαSu/IL-21 and IL-7/TF/IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al Transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.hum Gene Ther 2005, 16:457-72) and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allowed the formation and secretion of soluble IL-7/TF/IL-15, anti-CD 16scFv/IL-15RαSu/IL-21 protein complexes (called 7t15-16s21; FIGS. 75 and 76), which could be purified by affinity and other chromatographic methods based on anti-TF IgG1 antibodies.

Binding of 7t15-16s21 to CHO cells expressing human CD16b

CHO cells were transfected with pMC plasmid containing human CD16b and selected with 10 μg/mL blasticidin (blasticidin) for 10 days. CHO cells stably expressing CD16b were stained with 1.2 μg/mL of 7t15-16s21 with or 18t15-12s without anti-human CD16scFv as negative control and then stained with biotinylated anti-human tissue factor and PE-conjugated streptavidin. As shown in FIG. 77A, cells were stained with only anti-human CD16scFv containing 7t15-16s 21. As shown in FIG. 77B, 18t15-12s did not stain CHO cells expressing human CD 16B.

Detection of IL-15, IL-21 and IL-7 in 7t15-16s21 Using ELISA

96-well plates were coated with 100 μl (8 μg/mL) of anti-TF IgG1 in R5 (coating buffer) and incubated for 2 hours at Room Temperature (RT). Plates were washed 3 times and blocked with 100 μl 1% bsa in PBS. Serial dilutions of 7t15-16s21 (1:3 ratio) were added to wells and incubated for 60min at RT. After 3 washes, 50ng/mL of biotinylated anti-IL-15 antibody (BAM 247, andi organism), 500ng/mL of biotinylated anti-IL-21 antibody (13-7218-81, andi organism) or 500ng/mL of biotinylated anti-IL-7 antibody (506602, andi organism) was added to the wells and incubated for 60min at RT. Plates were washed 3 times and incubated with 0.25. Mu.g/mL HRP-SA (Jackson immunoresearch laboratory) at 100. Mu.L per well for 30min at RT, followed by washing 4 times and incubation with 100. Mu.L of ABTS for 2min at RT. Absorbance was read at 405 nm. As shown in FIGS. 78A-78C, IL-15, IL-21 and IL-7 domains in 7t15-16s21 were detected by individual antibodies.

IL-15 in 7t15-16s21 promotes proliferation of 32D beta cells containing IL-2R beta and a common gamma chain

To analyze the activity of IL-15 in 7t15-16s21, IL-15 activity of 7t15-16s21 was compared to recombinant IL-15 using 32D beta cells expressing IL2R beta and a common gamma chain, and its effect on promoting cell proliferation was evaluated. IL-15 dependent 32D beta cells were washed 5 times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells are seeded in the wells. Serial dilutions of 7t15-16s21 or IL-15 were added to cells (fig. 79). Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By adding 10. Mu.l WST1 to each well on day 3 and at CO ₂ Cell proliferation was detected by further incubation for 3 hours at 37℃in the incubator. Absorbance at 450nm was measured by analyzing the amount of formazan dye produced. As shown in FIG. 79, 7t15-16s21 and IL-15 promote 32D beta cell proliferation, and EC of 7t15-16s21 and IL-15 ₅₀ 172.2pM and 16.63pM, respectively.

Purification elution chromatography of 7t15-16s21 from anti-TF antibody affinity column

7t15-16s21 harvested from cell cultures were loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. The column was then washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. FIG. 80 is a graph showing the chromatographic profile after binding and elution of cell culture supernatants containing 7t15-16s21 protein on anti-TF antibody resin. As shown in FIG. 80, the anti-TF antibody affinity columns bind 7t15-16s21 containing TF. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Analytical Size Exclusion Chromatography (SEC) analysis of 7t15-16s21

For Size Exclusion Chromatography (SEC) analysis of 7t15-16s21, superdex200 Increate 10/300 GL gel filtration column (general electric medical group) connected to the AKTA Avant system (general electric medical group) was used. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7mL/min. Samples containing 7t15-16s21 in PBS were injected into a Superdex200 column using a capillary loop and analyzed by SEC. As shown in FIG. 81, SEC results indicated that 7t15-16s21 had two protein peaks.

Example 51: production and characterization of TGFRt15-16s21 fusion proteins

A fusion protein complex was generated comprising an anti-human CD16scFv/IL-15rαsu/IL21 and a tgfβ receptor II/TF/IL-15 fusion protein (figures 82 and 83). Human TGF-beta receptor II (Ile 24-Asp 159), tissue factor 219 and IL-15 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were prepared by ligating two TGF-beta receptor II sequences with a G4S (3) linker to produce a single-stranded form of TGF-beta receptor II, and then ligating directly to the N-terminal coding region of tissue factor 219, followed by ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of constructs comprising two TGF-beta receptor II linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequences (including signal peptide sequences) of the two TGF-beta receptor II/TF/IL-15 constructs are as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(two human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the TGF-beta receptor II/TF/IL-15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by ligating the anti-human CD16scFv sequence to the N-terminal coding region of the IL-15RαSu chain, followed by ligating to the N-terminal coding region of IL-21 synthesized by Jin Weizhi. The nucleic acid and protein sequences of constructs comprising an anti-human CD16scFv linked to the N-terminus of IL-15RαSu followed by the N-terminal coding region of IL-21 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the anti-CD 16scFv/IL-15RαSu/IL-21 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(anti-human CD16 scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence (including the signal peptide sequence) of the anti-CD 16scFv/IL-15RαSu/IL-21 construct is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(anti-human CD16 scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIR

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

The anti-CD 16scFv/IL-15RαSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al Transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005, 16:457-72) and the expression vectors transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble TGFR/TF/IL-15:CD16scFv/IL-15RαSu/IL-21 protein complex (called TGFRt15-16s 21) which can be purified by affinity based on anti-TF IgG1 and other chromatographic methods.

Interaction between TGFRt15-16s21 and CHO cells expressing human CD16b

CHO cells were transfected with human CD16b in pMC plasmid and selected with 10 μg/mL blasticidin for 10 days. Cells stably expressing CD16b were stained with 1.2. Mu.g/mL TGFRt15-16s21 with anti-human CD16scFv or 7t15-21s without anti-human CD16scFv as negative controls and stained with biotinylated anti-human tissue factor antibody and PE-conjugated streptavidin. As shown in FIGS. 84A and 84B, TGFRt15-16s21, which contained anti-human CD16scFv, showed positive binding, whereas 7t15-21s did not.

Effect of TGFRt15-16s21 on TGF-beta 1 Activity in HEK-Blue TGF beta cells

To assess the activity of TGFRt15-16s21 in TGFRt1, the effect of TGFRt15-16s21 on TGF 1 activity in HEK-Blue TGF beta cells was analyzed. HEK-Blue TGF beta cells (Invivogen) were washed twice with pre-warmed PBS and at 5X10 ⁵ Individual cells/mL were resuspended in test medium (DMEM, 10% heat inactivated FCS, 1x glutamine, 1x antibiotic-antifungal agent, and 2x glutamine). In a flat bottom 96-well plate, 50 μl of cells (2.5x10 ⁴ Individual cells/well) and then 50 μl of 0.1nM tgfβ1 (animate) was added. TGFRt15-16s21 or TGFR-Fc (Andi organism) prepared in 1:3 serial dilutions were then added to the plates to achieve a total volume of 200. Mu.L. After 24 hours incubation at 37 ℃, 40 μl of induced HEK-Blue tgfβ cell supernatant was added to 160 μl of pre-warmed QUANTI-Blue (Invivogen) in flat bottom 96 well plates and incubated for 1-3 hours at 37 ℃. Then using enzyme label instrument(Multiscan Sky) OD was measured at 620-655 nM. Calculation of IC for each protein sample using GraphPad Prism 7.04 ₅₀ . IC of TGFRt15-16s21 and TGFR-Fc ₅₀ 9127pM and 460.6pM, respectively. These results indicate that the TGFRt15-16s21 TGFβRII domain is capable of blocking TGFβ -1 activity in HEK-Blue TGFβ cells. IL-15 in TGFRt15-16s21 promotes proliferation of 32D beta cells containing IL-2R beta and common gamma chain

To analyze the activity of IL-15 in TGFRt15-16s21, IL-15 activity of TGFRt15-16s21 was compared with recombinant IL-15 using 32D beta cells expressing IL2R beta and a common gamma chain, and its effect on promoting cell proliferation was evaluated. IL-15 dependent 32D beta cells were washed 5 times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells are seeded in the wells. Serial dilutions of TGFRt15-16s21 or IL-15 were added to cells (fig. 86). Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By adding 10 μl of WST1 to each well on day 3 and at CO ₂ Cell proliferation was detected by further incubation for 3 hours at 37℃in the incubator. Absorbance at 450nm was measured by analyzing the amount of formazan dye produced. The data is shown in figure 85. As shown in FIG. 86, TGFRt15-16s21 and IL-15 promote 32D beta cell proliferation, and TGFRt15-16s21 and IL-15 EC ₅₀ 51298pM and 10.63pM, respectively.

Detection of IL-15, IL-21 and TGF-beta RII in TGFRt15-16s21 Using ELISA

96-well plates were coated with 100 μl (8 μg/mL) of anti-TF IgG1 in R5 (coating buffer) and incubated for 2 hours at Room Temperature (RT). Plates were washed 3 times and blocked with 100 μl 1% bsa in PBS. TGFRt15-16s21 was added serially diluted in a 1:3 ratio and incubated for 60min at RT. After three washes, 50ng/mL of biotinylated anti-IL-15 antibody (BAM 247, andi organism), 500ng/mL of biotinylated anti-IL-21 antibody (13-7218-81, andi organism) or 200ng/mL of biotinylated anti-TGF beta RII antibody (BAF 241, andi organism) was applied per well and incubated for 60min at RT. After three washes, incubation with 0.25. Mu.g/mL HRP-SA (Jackson ImmunoResearch at 100. Mu.L per well for 30min at RT followed by 4 washes and with 100. Mu.L ABTS for 2min at RT. Absorbance was read at 405 nm. IL-15, IL-21 and TGFβRII domains in TGFRt15-16s21 were detected by the corresponding antibodies as shown in FIGS. 87A-87C.

Purification elution chromatography of TGFRt15-16s21 using anti-TF antibody affinity column

TGFRt15-16s21 harvested from cell cultures was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized samples were then buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. As shown in fig. 88, the anti-TF antibody affinity column binds to TGFRt15-16s21, which contains tissue factor as a fusion partner. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Reduced SDS-PAGE of TGFRt15-16s21

To determine the purity and molecular weight of the TGFRt15-16s21 protein, protein samples purified with anti-TF antibody affinity columns were analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) under reducing conditions. After electrophoresis, the gel was stained with instant blue for about 30min, and then decolorized in purified water overnight.

To verify that the TGFRt15-16s21 protein undergoes glycosylation post-translationally in CHO cells, deglycosylation experiments were performed using the protein deglycosylation mixture II kit from the new england biology laboratory according to the manufacturer's instructions. FIG. 89 shows the results of a reduction SDS-PAGE analysis of the samples in the non-deglycosylated (red outline lane 1) and deglycosylated (yellow outline lane 2) state. The results indicate that the TGFRt15-16s21 protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified samples showed the expected molecular weights (69 kDa and 48 kDa) in a reduced SDS gel. Lane M was loaded with 10. Mu.L of SeeBlue Plus2 pre-stain.

Example 52: generation and characterization of 7t15-7s fusion proteins

A fusion protein complex was generated comprising IL-7/TF/IL-15 and IL-7/IL-15rαsu fusion proteins (fig. 90 and 91). Human IL-7, tissue factor 219 and IL-15 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were made that ligate the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by ligation to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of constructs comprising IL-7 linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the 7t15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL 7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the 7t15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL 7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by ligating the IL-7 sequence to the N-terminal coding region of the IL-15RαSu chain synthesized by Jin Weizhi. The nucleic acid and protein sequences of constructs comprising IL-7 linked to the N-terminus of the IL-15RαSu chain are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the 7s construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL 7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence (including the leader sequence) of the 7s fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL 7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

IL-7/TF/IL-15 and IL-7/IL-15RαSu constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu Y, dudley ME, zheng Z, robbins PF, li Y et al, transfer of aTCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.hum Gene Ther 2005; 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble IL-7/TF/IL-15:IL-7/IL-15RαSu protein complexes called 7t15-7s, which can be purified by anti-TF antibody IgG1 affinity and other chromatographic methods. Purification elution chromatography of 7t15-7s using anti-TF antibody affinity column

7t15-7s from cell cultures were loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized samples were then buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. As shown in FIG. 92, the anti-TF antibody affinity column binds 7t15-7s containing Tissue Factor (TF) as a fusion partner. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min. Immunostimulation of 7t15-7s in C57BL/6 mice

7t15-7s is a multi-chain polypeptide (type A multi-chain polypeptide described herein) comprising a first polypeptide (7 t 15) that is a soluble fusion of human IL-7, human tissue factor 219 fragment and human IL-15, and a second polypeptide (7 s) that is a soluble fusion of the sushi domain of the alpha chain of human IL-7 and human IL-15 receptor.

CHO cells were co-transfected with IL7-TF-IL-15 (7 t 15) and IL7-IL-15Ra sushi domain (7 s) vectors. The 7t15-7s complex was purified from the transfected CHO cell culture supernatant. The IL-7, IL-15 and Tissue Factor (TF) components of the complexes were demonstrated by ELISA as shown in FIG. 93. Humanized anti-TF monoclonal antibodies (anti-TF IgG 1) were used as capture antibodies for the determination of TF in 7t15-7s, and biotinylated anti-human IL-15 antibodies (animate) and biotinylated anti-human IL-7 antibodies (animate) were used as detection antibodies for the detection of IL-15 and IL-7 in 7t15-7s, respectively, followed by peroxidase-conjugated streptavidin (jackson immunoresearch laboratory) and ABTS substrates (surmod IVD, inc.).

7t15-7s was subcutaneously injected into C57BL/6 mice at 10mg/kg to determine the immunostimulatory activity of 7t15-7s in vivo. C57BL/6 mice treated subcutaneously with PBS were used as controls. Spleens of mice were collected and weighed on day 4 post-treatment. Single spleen cell suspensions were prepared and stained with fluorochrome-conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 antibodies, and CD4 was analyzed by flow cytometry ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells. The results showed that 7t15-7s effectively expanded spleen cells based on spleen weight (FIG. 94A), and specifically CD8 ⁺ The percentage of T cells and NK cells was higher than the control treated mice (fig. 94B).

Example 53: production and characterization of TGFRt15-TGFRs fusion proteins

A fusion protein complex was generated comprising tgfβ receptor II/IL-15rαsu and tgfβ receptor II/TF/IL-15 fusion proteins (fig. 95 and 96). Human TGF-beta receptor II (Ile 24-Asp 159), tissue factor 219 and IL-15 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were prepared by ligating two TGF-beta receptor II sequences with a G4S (3) linker to produce a single-stranded form of TGF-beta receptor II, and then ligating directly to the N-terminal coding region of tissue factor 219, followed by ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of constructs comprising two TGF-beta receptor II linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequences (including signal peptide sequences) of the two TGF-beta receptor II/TF/IL-15 constructs are as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(two human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the TGF-beta receptor II/TF/IL-15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly linking two tgfβ receptor II to the IL-15rαsu chain synthesized by Jin Weizhi. The nucleic acid and protein sequences comprising the construct with TGF-beta receptor II linked to the N-terminus of IL-15RαSu are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGF-beta receptor II/IL-15RαSu construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(two human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequences (including the signal peptide sequences) of the two TGF-beta receptor II/IL-15RαSu constructs are as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(two human TGF-beta receptor II extracellular domains)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

TGF-beta R/IL-15RαSu and TGF-beta R/TF/IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu Y, dudley ME, zheng Z, robbins PF, li Y et al, transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.hum Gene Ther 2005; 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble TGF-beta R/TF/IL-15 TGF-beta R/IL-15R alpha Su protein complexes (known as TGFRt 15-TGFRs), which can be purified by anti-TF IgG1 affinity and other chromatographic methods.

Effect of TGFRt15-TGFRs on TGF-beta 1 Activity in HEK-Blue TGF beta cells

To assess the activity of TGFRt15-TGFRs in TGFRt RII, the effect of TGFRt15-16s21 on TGF beta 1 activity in HEK-Blue TGF beta cells was analyzed. HEK-Blue TGF beta cells (Invivogen) were washed twice with pre-warmed PBS and at 5X10 ⁵ Individual cells/mL were resuspended in test medium (DMEM, 10% heat inactivated FCS, 1x glutamine, 1x antibiotic-antifungal agent, and 2x glutamine). In a flat bottom 96-well plate, 50 μl of cells (2.5x10 ⁴ Individual cells/well) and then 50 μl of 0.1nM tgfβ1 (animate) was added. TGFRt15-16s21 or TGFR-Fc (Andi organism) prepared in 1:3 serial dilutions were then added to the plates to achieve a total volume of 200. Mu.L. After 24 hours incubation at 37 ℃, 40 μl of induced HEK-Blue tgfβ cell supernatant was added to 160 μl of pre-warmed QUANTI-Blue (infljie) in flat bottom 96 well plates and incubated for 1-3 hours at 37 ℃. OD values were then determined using a microplate reader (Multiscan Sky) at 620-655nM (fig. 97). Calculation of IC for each protein sample using GraphPad Prism 7.04 ₅₀ . IC of TGFRt15-TGFRs and TGFR-Fc ₅₀ 216.9pM and 460.6pM, respectively. These results indicate that the TGFRt15-TGFRs TGFβRII domain is capable of blocking TGFβ1 activity in HEK-Blue TGFβ cells.

IL-15 in TGFRt15-TGFRs promotes proliferation of 32D beta cells containing IL-2R beta and common gamma chain

To evaluate the activity of IL-15 in TGFRt15-TGFRs, IL-15 activity of TGFRt15-TGFRs was compared to recombinant IL-15 using 32D beta cells expressing IL2R beta and a common gamma chain, and its effect on promoting cell proliferation was evaluated. IL-15 dependent 32D beta cells were washed 5 times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells are seeded in the wells. Serial dilutions of TGFRt15-TGFRs or IL-15 were added to cells (fig. 98). Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By following each on day 3Add 10. Mu.L WST1 to the well and add CO ₂ Cell proliferation was detected by further incubation for 3 hours at 37℃in the incubator. Absorbance at 450nm was measured by analyzing the amount of formazan dye produced. As shown in FIG. 98, TGFRt15-TGFRs and IL-15 promote 32D beta cell proliferation, TGFRt15-16s21 and IL-15 EC ₅₀ 1901pM and 10.63pM, respectively.

Detection of IL-15 and TGFβRII domains in TGFRt15-TGFRs using ELISA with corresponding antibodies

96-well plates were coated with 100 μl (8 μg/mL) of anti-TF IgG1 in R5 (coating buffer) and incubated for 2 hours at Room Temperature (RT). Plates were washed 3 times and blocked with 100 μl 1% bsa in PBS. TGFRt15-TGFRs was added in 1:3 serial dilutions and incubated for 60 minutes at RT. After 3 washes, 50ng/mL of biotinylated anti-IL-15 antibody (BAM 247, andi organism) or 200ng/mL of biotinylated anti-TGFbRII antibody (BAF 241, andi organism) was added to the wells and incubated for 60min at RT. Plates were then washed 3 times and 0.25 μg/mL HRP-SA (jackson immunoresearch laboratory) was added at 100 μl per well and incubated for 30min at RT, followed by washing 4 times and incubation with 100 μl ABTS for 2min at RT. The absorbance at 405nm was read. IL-15 and TGFβRII domains in TGFRt15-TGFRs were detected by individual antibodies as shown in FIGS. 99A and 99B.

Purification elution chromatography of TGFRt15-TGFRs from anti-TF antibody affinity column

TGFRt15-TGFRs collected from cell cultures were loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized samples were then buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. As shown in figure 100, the anti-TF antibody affinity column binds to TGFRt15-TGFRs containing TF as a fusion partner. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Analytical Size Exclusion Chromatography (SEC) analysis of TGFRt15-TGFRs

Superdex200 Increate 10/300GL gel filtration column (from general electric healthcare group) was connected to the AKTA Avant system (from general electric healthcare group). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7mL/min. Samples containing TGFRt15-TGFRs in PBS were injected into Superdex200 column using capillary loop and analyzed by SEC. SEC chromatograms of the samples are shown in figure 101. SEC results showed that there were four protein peaks in TGFRt 15-TGFRs.

Reduced SDS-PAGE analysis of TGFRt15-TGFRs

To determine the purity and molecular weight of the TGFRt15-TGFRs protein, protein samples purified with anti-TF antibody affinity columns were analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) under reducing conditions. After electrophoresis, the gel was stained with instant blue for about 30min, and then decolorized in purified water overnight.

To verify that the TGFRt15-TGFRs proteins undergo glycosylation post-translationally in CHO cells, deglycosylation experiments were performed using the protein deglycosylation mixture II kit from the new england biology laboratory and the manufacturer's instructions. FIG. 102 shows the SDS-PAGE analysis of samples in the non-deglycosylated (red outline lane 1) and deglycosylated (yellow outline lane 2) states. The results indicate that the TGFRt15-TGFRs protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified samples showed the expected molecular weights (69 kDa and 39 kDa) in a reduced SDS gel. Lane M was loaded with 10ul SeeBlue Plus2 pre-staining standard.

Immunostimulatory Activity of TGFRt15-TGFRs in C57BL/6 mice

TGFRt15-TGFRs are multi-chain polypeptides (type a multi-chain polypeptides described herein) comprising a first polypeptide that is a soluble fusion of two tgfbetarii domain human tissue factor 219 fragments and human IL-15 and a second polypeptide that is a soluble fusion of two tgfbetarii domains and sushi domain of the human IL-15 receptor alpha chain.

Wild type C57BL/6 mice were treated subcutaneously with control solutions or with TGFRt15-TGFRs at doses of 0.3mg/kg, 1mg/kg, 3mg/kg or 10 mg/kg. Four days after treatment, spleen weight and the percentage of various immune cell types present in the spleen were assessed. As shown in fig. 103A, spleen weight of mice treated with TGFRt15-TGFRs increased with increasing dose of TGFRt 15-TGFRs. Furthermore, mice treated with 1mg/kg, 3mg/kg and 10mg/kg of TGFRt15-TGFRs, respectively, had higher spleen weights than control solution treated mice. In addition, the presence of CD4 in the spleen of control-treated and TGFRt 15-TGFRs-treated mice was assessed ⁺ T cells, CD8 ⁺ T cells, NK cells and CD19 ⁺ Percentage of B cells. As shown in FIG. 103B, CD8 in spleen of mice treated with TGFRt15-TGFRs ⁺ The percentage of both T cells and NK cells increased with increasing TGFRt15-TGFRs dose. Specifically, CD8 in mice treated with 0.3mg/kg, 3mg/kg and 10mg/kg TGFRt15-TGFRs compared to control treated mice ⁺ The percentage of T cells was higher and the percentage of NK cells in mice treated with 0.3mg/kg, 1mg/kg, 3mg/kg and 10mg/kg TGFRt15-TGFRs was higher than in control treated mice. These results indicate that TGFRt15-TGFRs are able to stimulate immune cells, especially CD8, in the spleen ⁺ T cells and NK cells.

The pharmacokinetics of the TGFRt15-TGFRs molecules were evaluated in wild-type C57BL/6 mice. Mice were treated subcutaneously with a dose of 3mg/kg of TGFRt 15-TGFRs. Mouse blood was discharged from the tail vein at each time point, and serum was prepared. TGFRt15-TGFRs concentrations in mouse serum were determined by ELISA (capture: anti-human tissue factor antibody; detection: biotinylated anti-human TGF-beta receptor antibody followed by peroxidase-conjugated streptavidin and ABTS substrate). The results showed that the half-life of TGFRt15-TGFRs in C57BL/6 mice was 12.66 hours.

Preparation of mouse spleen cells for evaluation of TGFRt15-TGFRs in mice over timeIs a potent immunostimulant activity of (a). As shown in fig. 104A, spleen weight of mice treated with TGFRt15-TGFRs increased 48 hours after treatment and continued to increase over time. In addition, the presence of CD4 in the spleen of control-treated and TGFRt 15-TGFRs-treated mice was assessed ⁺ T cells, CD8 ⁺ T cells, NK cells and CD19 ⁺ Percentage of B cells. As shown in FIG. 104B, CD8 was found 48 hours after the treatment in the spleen of the mice treated with TGFRt15-TGFRs ⁺ The percentage of both T cells and NK cells increased and increased over time after single dose treatment. These results further indicate that TGFRt15-TGFRs are able to stimulate immune cells, especially CD8, in the spleen ⁺ T cells and NK cells.

In addition, in splenocytes isolated from mice after a single dose (3 mg/kg) of TGFRt15-TGFRs, the dynamic proliferation of immune cells based on expression of splenocyte Ki67 and the cytotoxic potential based on expression of granzyme B were evaluated. As shown in fig. 105A and 105B, in spleen of mice treated with TGFRt15-TGFRs, ki67 and granzyme B expression of NK cells increased and CD8 at 24 hours after treatment ⁺ Expression of Ki67 and granzyme B by T cells and NK cells increased at both 48 hours and later time points after single dose treatment. These results indicate that TGFRt15-TGFRs not only increases CD8 ⁺ T cells and NK cells, and enhance the cytotoxicity of these cells. Single dose treatment of TGFRt15-TGFRs causes CD8 ⁺ T cells and NK cells proliferate for at least 4 days.

Spleen cells from mice treated with TGFRt15-TGFRs were also evaluated for cytotoxicity against tumor cells. Small Moloney (Moloney) leukemia cells (Yac-1) were labeled with CellTrace violet and used as tumor target cells. At various time points after treatment, spleen cells were prepared from the spleen of mice treated with TGFRt15-TGFRs (3 mg/kg) and used as effector cells. Target cells were mixed with effector cells at E: T ratio = 10:1 and incubated for 20 hours at 37 ℃. Target cell viability was assessed by analysis of propidium iodide positive, violet labeled Yac-1 cells using flow cytometry. The percent inhibition of Yac-1 tumors was calculated using the following formula: (1- [ Yac-1 viable cell count in the test sample ]/[ Yac-1 viable cell count in the sample without spleen cells ]) x100. As shown in FIG. 106, splenocytes from TGFRt15-TGFRs treated mice were more cytotoxic to Yac-1 cells than control mice.

Tumor size analysis in response to chemotherapy and/or TGFRt15-TGFRs

Pancreatic cancer cells (SW 1990,CRL-2172) was injected subcutaneously (s.c.) into C57BL/6scid mice (Jackson laboratories, 001913,2x 10) ⁶ Individual cells/mouse in 100 μl HBSS) to establish a pancreatic cancer mouse model. Two weeks after tumor cell injection, chemotherapy with albumin paclitaxel (Celgene, 68817-134,5mg/kg, i.p.) in combination with gemcitabine (sigma aldrich, G6423, 40mg/kg, i.p.) followed by immunotherapy with TGFRt15-TGFRs (3 mg/kg, s.c.) within 2 days was started in these mice. The above procedure is considered as one treatment cycle and is repeated for another 3 cycles (1 cycle/week). The control group was set up as SW1990 injected mice that received PBS, chemotherapy (gemcitabine and albumin paclitaxel) or TGFRt15-TGFRs alone. Tumor size was measured and recorded every other day throughout the treatment cycle for each animal until the 2 month experiment was terminated after SW1990 cell injection. Tumor volume measurements were analyzed by group and the results showed that the tumors of animals receiving chemotherapy in combination with TGFRt15-TGFRs were significantly smaller compared to PBS group, whereas neither chemotherapy nor TGFRt15-TGFRs therapy alone performed as well as the combination (fig. 107).

In vitro aging B16F10 melanoma model

Next, activated mouse NK cells were evaluated to kill senescent B16F10 melanoma cells in vitro. B16F10 senescent cells (B16F 10-SNC) were labeled with CellTrace violet and incubated with 2T2 activated mouse NK cells (isolated from spleens of C57BL/6 mice injected with TGFRt15-TGFRs 10mg/kg for 4 days) for 16 hours at different E:T ratios in vitro. Cells were trypsinized, washed and resuspended in complete medium containing Propidium Iodide (PI) solution. Cytotoxicity was assessed by flow cytometry (fig. 108).

Example 54:7t15-21s137L (long version) fusion protein production and characterization

A fusion protein complex was generated comprising IL-21/IL-15RαSu/CD137L and IL-7/TF/IL-15 fusion proteins (FIGS. 109 and 110). Specifically, constructs were made as follows: the IL-7 sequence is linked to the N-terminal coding region of tissue factor 219, which is in turn linked to the N-terminal coding region of IL-15. The nucleic acid and protein sequences of constructs comprising IL-7 linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the 7t15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL 7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the 7t15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL 7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid and protein sequences of 21s137L are shown below. The nucleic acid sequence (including the signal peptide sequence) of the 21s137L construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S) 3 Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human CD 137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including the leader sequence) of the 21s137L fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

((G4S) 3 Joint)

GGGGSGGGGSGGGGS

(human CD 137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

IL-21/IL-15RαSu/CD137L and IL-7/TF/IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al, transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005, 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble IL-7/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complexes (referred to as 7t15-21s 137L), which can be purified by anti-TF antibody IgG1 affinity and other chromatographic methods.

Purification elution chromatography using 7t15-21s137L of anti-TF antibody affinity column

7t15-21s137L from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized samples were then buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. As shown in FIG. 111, the anti-TF antibody affinity column binds 7t15-21s137L containing TF as a fusion partner. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. All steps were at a flow rate of 4mL/min, except for the elution step, which was 2mL/min. FIG. 112 shows analytical SEC profiles of 7t15-21s 137L.

Example 55: generation and characterization of 7t15-21s137L (short version) fusion proteins

A fusion protein complex is generated comprising IL-21/IL-15RαSu/CD137L and IL-7/TF/IL-15 fusion proteins. Specifically, constructs were made that ligate the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by ligation to the N-terminal coding region of IL-15. The nucleic acid and protein sequences of constructs comprising IL-7 linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the 7t15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL 7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the 7t15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL 7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid and protein sequences of 21s137L (short version) are shown below. The nucleic acid sequence (including the signal peptide sequence) of the 21s137L (short version) construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S) 3 Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human CD137 ligand short version)

GATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC

The amino acid sequence (including the signal peptide sequence) of the 21s137L (short version) construct is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

((G4S) 3 Joint)

GGGGSGGGGSGGGGS

(human CD137 ligand short version)

DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI

IL-21/IL-15RαSu/CD137L (short version) and IL-7/TF/IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al, transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble IL-7/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complexes (called 7t15-21s137L (short version)), which can be purified by anti-TF antibody IgG1 affinity and other chromatographic methods.

Binding of 7t15-21s137L (short version) to CD137 (4.1 BB)

On day 1, 96-well plates were coated with 100 μl (2.5 μg/mL) of R5 (coating buffer) or R5 alone containing GAH IgG Fc (G-102-C, animate) and incubated overnight at 4 ℃. On day 2, the plates were washed three times and blocked with 300 μl of 1% bsa in PBS for 2 hours at 37 ℃. 10ng/mL of 4.1BB/Fc (838-4B, andi organism) was added at 100. Mu.L/well and incubated for 2 hours at RT. After three washes, 7t15-21s137L or 7t15-21s was serially diluted at a ratio of 1/3 (starting from 10 nM) and incubated overnight at 4 ℃. On day 3, after 3 washes, 300ng/mL of biotinylated anti-hTF antibody (BAF 2339, animate) was added at 100 μl per well and incubated for 2 hours at RT. Plates were then washed three times and incubated with 0.25. Mu.g/mL HRP-SA (Jackson immunization study) for 30min at 100. Mu.L per well, followed by 3 times and 2min at RT with 100. Mu.L of ABTS. Absorbance was read at 405 nm. As shown in FIG. 113, 7t15-21s137L (short version) shows significant interactions with 4.1BB/Fc (blue line) compared to 7t15-21 s.

IL-15, IL-21 and IL-7 were detected in 7t15-21s137L (short version) by ELISA

The 96-well plates were coated with 100. Mu.L of R5 (coating buffer) containing (8. Mu.g/mL) anti-TF antibody IgG1 and incubated for 2 hours at RT. Plates were washed 3 times and blocked with 100 μl of PBS containing 1% bsa. 7t15-21s137L (short version) serially diluted in a 1:3 ratio was added and incubated for 60 minutes at RT. After three washes, 50ng/mL of biotinylated anti-IL-15 antibody (BAM 247, andi organism), 500ng/mL of biotinylated anti-IL 21 antibody (13-7218-81, andi organism) or 500ng/mL of biotinylated anti-IL 7 antibody (506602, andi organism) was added to the wells and incubated for 60 minutes at RT. Three washes and incubation with 0.25. Mu.g/mL HRP-SA (Jackson immunization study) at 100. Mu.L per well for 30 min at RT followed by four washes and incubation with 100. Mu.L ABTS for 2 min at RT. Absorbance was read at 405 nm. As shown in FIGS. 114A-114C, IL-15, IL-21 and IL-7 domains in 7t15-21s137L (short version) were detected by the corresponding antibodies.

IL-15 in 7t15-1s137L (short version) promotes proliferation of CTLL2 cells containing IL2Rαβγ

To assess the activity of IL-15 in 7t15-21s137L (short version), 7t15-21s137L (short version) was compared with recombinant IL-15 in promoting proliferation of CTLL2 cells expressing IL2Rαβγ. IL-15 dependent CTLL2 cells were washed 5 times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells were seeded into the wells. Serial dilutions of 7t15-21s137L (short version) or IL-15 were added to cells (fig. 115). Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By adding 10 μl of WST1 to each well on day 3, and at CO ₂ Cell proliferation was detected by further incubation for 3 hours at 37℃in the incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in FIG. 115, 7t15-21s137L (short version) and IL-15 promote CTLL2 cell proliferation. EC of 7t15-21s137L (short version) and IL-15 ₅₀ 55.91pM and 6.22pM, respectively.

IL-21 in 7t15-1s137L (short version) promotes proliferation of B9 cells containing IL21R

To evaluate IL-21 activity of 7t15-21s137L (short version), 7t15-21s137L (short version) was compared to recombinant IL-21 in promoting proliferation of B9 cells expressing IL-21R. B9 cells containing IL-21R were washed 5 times with RPMI-10% FBS and 1X10 ⁴ Individual cells/wells were seeded into the wells. Serial dilutions of 7t15-21s137L (short version) or IL-21 were added to cells (fig. 116). In CO ₂ Cells were incubated in the incubator at 37℃for 5 days. By adding 10 μl of WST1 to each well on day 5, and at CO ₂ At 37 ℃ in an incubatorThe cells were further cultured for 4 hours to examine proliferation. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in FIG. 116, 7t15-21s137L (short version) and IL-21 promote B9 cell proliferation. EC of 7t15-21s137L (short version) and IL-21 ₅₀ 104.1nM and 72.55nM, respectively.

Example 56: generation and characterization of 7t15-TGFRs fusion proteins

A fusion protein complex was generated comprising tgfβ receptor II/IL-15rαsu and IL-7/TF/IL-15 fusion proteins (fig. 117 and 118). Human TGF-beta receptor II (Ile 24-Asp 159), tissue factor 219, IL-15 and IL-7 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were made that ligate the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by ligation to the N-terminal coding region of IL-15. The nucleic acid and protein sequences of constructs comprising IL-7 linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the 7t15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL 7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the 7t15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL 7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly linking two tgfβ receptor II to the IL-15rαsu chain synthesized by Jin Weizhi. The nucleic acid and protein sequences comprising the construct with TGF-beta receptor II linked to the N-terminus of IL-15RαSu are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRs construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The amino acid sequence (including the leader sequence) of the TGFRs fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIR

Effect of 7t15-TGFRs on TGF-beta 1 Activity in HEK-Blue TGF-beta cells

To assess the activity of TGF-beta R in 7t15-TGFRs, the effect of 7t15-TGFRs on TGF-beta 1 activity in HEK-Blue TGF-beta cells was analyzed. HEK-Blue TGF beta cells (Invivogen) were washed twice with pre-warmed PBS and at 5X10 ⁵ Individual cells/mL were resuspended in test medium (DMEM, 10% heat inactivated FCS, 1x glutamine, 1x antibiotic-antifungal agent, and 2x glutamine). In a flat bottom 96-well plate, 50 μl of cells (2.5x10 ⁴ Individual cells/well) and then 50 μl of 0.1nM tgfβ1 (animate) was added. 7t15-TGFRs or TGFR-Fc (Andi organism) prepared in 1:3 serial dilutions were then added to the plates to achieve a total volume of 200. Mu.L. After 24 hours incubation at 37 ℃, 40 μl of induced HEK-Blue tgfβ cell supernatant was added to 160 μl of pre-warmed QUANTI-Blue (Invivogen) in flat bottom 96 well plates and incubated for 1-3 hours at 37 ℃. OD values were then determined using a microplate reader (Multiscan Sky) at 620-655 nM. The data is shown in figure 119. Calculation of IC for each protein sample using GraphPad Prism 7.04 ₅₀ . IC of 7t15-TGFRs and TGFR-Fc ₅₀ 1142pM and 558.6pM, respectively. These results indicate that TGF-. Beta.R in 7t15-TGFRs is capable of blocking TGF-. Beta.1 activity in HEK-Blue TGF-. Beta.cells. Detection of IL-15, TGF-beta RII and IL-7 in 7t15-TGFRs by ELISA

The 96-well plates were coated with 100. Mu.L of R5 (coating buffer) containing (8. Mu.g/mL) anti-TF antibody IgG1 and incubated for 2 hours at Room Temperature (RT). Plates were washed three times and blocked with 100 μl of PBS containing 1% bsa. Serial dilutions of 7t15-TGFRs (1:3 ratio) were added and incubated for 60min at RT. After 3 washes, 50ng/mL of biotinylated anti-IL-15 antibody (BAM 247, andi organism), 200ng/mL of biotinylated anti-TGFbRII antibody (BAF 241, andi organism) or 500ng/mL of biotinylated anti-IL-7 antibody (506602, andi organism) was added and incubated for 60min at RT. After three washes, incubation with 0.25. Mu.g/mL HRP-SA (Jackson immunization study) was performed for 30min at 100. Mu.L per well at RT, followed by 4 washes and 2min at RT with 100. Mu.L ABTS. Absorbance was read at 405 nm. IL-15, TGFR and IL-7 in 7t15-TGFRs were detected by the corresponding antibodies as shown in FIGS. 120A-120C.

IL-15 in 7t15-TGFRs promotes proliferation of 32D beta cells containing IL-2R beta and a common gamma chain

To assess the activity of IL-15 in 7t15-TGFRs, 7t15-TGFRs were compared to recombinant IL-15 using 32D beta cells expressing IL2R beta and a common gamma chain, and their effect on promoting cell proliferation was assessed. IL-15 dependent 32D beta cells were washed 5 times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells are seeded in the wells. Serial dilutions of 7t15-TGFRs or IL-15 were added to cells (fig. 121). Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By adding 10 μl of WST1 to each well on day 3 and at CO ₂ Cell proliferation was detected by further incubation for 3 hours at 37℃in the incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in FIG. 121, 7t15-TGFRs and IL-15 promote 32D beta cell proliferation, and EC of 7t15-TGFRs and IL-15 ₅₀ 126nM and 16.63pM, respectively. Purification elution chromatography of 7t15-TGFRs using anti-TF antibody affinity column

7t15-TGFRs harvested from cell cultures were loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (ph 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized samples were then buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. As shown in figure 122, the anti-TF antibody affinity column bound to 7t15-TGFRs containing TF as a fusion partner for 7t 15-TGFRs. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Reduced SDS-PAGE analysis of 7t15-TGFRs

To determine the purity and molecular weight of the protein, 7t15-TGFRs protein samples purified with anti-TF antibody affinity columns were analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) under reducing conditions. After electrophoresis, the gel was stained with instant blue for about 30min, and then decolorized in purified water overnight.

To verify that 7t15-TGFRs proteins were post-translationally glycosylated in CHO cells, deglycosylation experiments were performed using the protein deglycosylation mixture II kit from the new england biology laboratory and the manufacturer's instructions. FIG. 123 shows the SDS-PAGE analysis of the samples in the non-deglycosylated (red outline lane 1) and deglycosylated (yellow outline lane 2) state. These results indicate that the protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified samples showed the expected molecular weights (55 kDa and 39 kDa) in a reduced SDS gel. Lane M was loaded with 10ul SeeBlue Plus2 pre-staining standard.

Characterization of 7t15-TGFRs

7t15-TGFRs are multi-chain polypeptides (type A multi-chain polypeptides described herein) comprising a first polypeptide (7 t 15) that is a soluble fusion of human IL-7, human tissue factor 219 fragment and human IL-15, and a second polypeptide (TGFRs) that is a soluble fusion of two TGF-beta RII domains that are single chain and the alpha chain sushi domain of the human IL-15 receptor.

CHO cells were co-transfected with 7t15 and TGFRs vector. The 7t15-TGFRs complex was purified from the transfected CHO cell culture supernatant. As shown in FIG. 124, IL-7, IL-15, TGF-beta receptor and Tissue Factor (TF) components in the complexes were demonstrated by ELISA. TF in 7t15-TGFRs was determined using humanized anti-TF antibody monoclonal antibody (anti-TF antibody IgG 1) as a capture antibody, and IL-7, IL-15 and TGF-beta receptors in 7t15-TGFRs were determined using biotinylated antibodies against human IL-15 antibody (Andi organism), human IL-7 (Bai jin Biotechnology), anti-TGF-beta receptor (Andi organism) as detection antibodies, respectively. The bound biotinylated antibodies were then detected using peroxidase-conjugated streptavidin (jackson immunoresearch laboratory) and ABTS substrate (surmod IVD, inc.). The results were analyzed by ELISA (fig. 124).

In vivo characterization of 7t15-TGFRs in C57BL/6 mice

To determine the immunostimulatory activity of 7t15-TGFRs in vivo, C57BL/6 mice were treated subcutaneously with control solution (PBS) or with 0.3, 1, 3 and 10mg/kg of 7t 15-TGFRs. Treated mice were euthanized. Spleens of mice were collected and weighed on day 4 post-treatment. Single spleen cell suspensions were prepared and stained with fluorochrome-conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 antibodies, and CD4 was analyzed by flow cytometry ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells. The results showed that 7t15-TGFRs were effective in expanding spleen cells, especially at 1-10mg/kg based on spleen weight (FIG. 125A). CD8 compared to control treated mice at all tested doses ⁺ The percentages of T cells and NK cells were higher (fig. 125B).

CD44 expression by CD4+ and CD8+ T cells

IL-15 is known to induce CD44 expression on T cells and the development of memory T cells. Assessment of CD4 in 7t15-TGFRs treated mice ⁺ And CD8 ⁺ CD44 expression by T cells. C57BL/6 mice were treated subcutaneously with 7t 15-TGFRs. Spleen cells were stained with fluorochrome-conjugated anti-CD 4, anti-CD 8 and anti-CD 44 monoclonal antibodies against immune cell subsets. Analysis of CD4 of total CD4+ T cells by flow cytometry ⁺ CD44 ^{High height} Percentage of T cells and Total CD8 ⁺ CD8 of T cells ⁺ CD44 ^{High height} Percentage of T cells. As shown in figures 126A and 126B, 7t15-TGFRs significantly activated CD4 ⁺ And CD8 ⁺ T cells differentiate into memory T cells.

In addition, the 7t15-TGFRs induced dynamic proliferation of spleen cells based on Ki67 expressed immune cells and the cytotoxic potential of spleen cells based on granzyme B expression after single dose treatment of mice was also assessed. C57BL/6 mice were treated subcutaneously with 3mg/kg of 7t 15-TGFRs. Treated mice were euthanized and spleen cells were prepared. Prepared spleen cells were conjugated with fluorochromes for anti-CD 4, anti-CD 8 and anti-CD -NK1.1 (NK) antibody was stained against immune cell subpopulations and then intracellular staining with anti-Ki 67 antibody against cell proliferation and intracellular staining with anti-granzyme B antibody against cytotoxic markers. The Ki67 and granzyme B corresponding immune cell subpopulations were analyzed for Mean Fluorescence Intensity (MFI) by flow cytometry. As shown in FIGS. 127A and 127B, CD8 in the spleen of mice treated with 7t15-TGFRs compared to PBS control treatment ⁺ The expression of both Ki67 and granzyme B by T cells and NK cells was increased. These results indicate that 7t15-TGFRs not only increases CD8 ⁺ T cells and NK cells, and enhance the potential cytotoxicity of these cells.

In addition, mouse spleen cells were also evaluated for cytotoxicity to tumor cells. Mouse Yac-1 cells were labeled with CellTrace violet and used as tumor target cells. Spleen cells were prepared from 7t15-TGFRs treated mice and used as effector cells. Target cells were mixed with effector cells in RPMI-10 medium with or without 100nM of 7T15-TGFRs at E: T ratio = 10:1 and incubated for 20 hours at 37 ℃. Target Yac-1 cell inhibition was assessed by analyzing ionomarked living Yac-1 cells using flow cytometry. The percentage of Yac-1 inhibition was calculated using the following formula: (1-Yac-1 viable cell count in the test sample/Yac-1 viable cell count in the sample without splenocytes) x100. As shown in FIG. 128, 7t15-TGFRs treated mouse spleen cells were more cytotoxic to Yac-1 cells than control mouse spleen cells, and the addition of 7t15-TGFRs further enhanced spleen cell cytotoxicity to Yac-1 target cells during cytotoxicity assays.

Example 57: production and characterization of TGFRt15-21s137L fusion protein

A fusion protein complex was generated comprising IL-21/IL-15rαsu/CD137L and tgfβ receptor II/TF/IL-15 fusion proteins (fig. 129 and 130). Human TGF-beta receptor II (Ile 24-Asp 159), tissue factor 219 and IL-15 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were prepared by ligating two TGF-beta receptor II sequences to a G4S (3) linker to produce a single-stranded form of TGF-beta receptor II, and then directly to the N-terminal coding region of tissue factor 219, followed by ligation to the N-terminal coding region of IL-15.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRt15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the TGFRt15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid and protein sequences of 21s137L are shown below. The nucleic acid sequence (including the signal peptide sequence) of the 21s137L construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S) 3 Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human CD 137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including the leader sequence) of the 21s137L fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTT PSLKCIR

((G4S) 3 Joint)

GGGGSGGGGSGGGGS

(human CD 137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

IL-21/IL-15RαSu/CD137L and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al, transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72), and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble TGFR/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complex (called TGFRt15-21s 137L) which can be purified by anti-TF antibody IgG1 affinity and other chromatographic methods.

Purification elution chromatography of TGFRt15-21s137L using anti-TF antibody affinity column

TGFRt15-21s137L from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized samples were then buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. As shown in FIG. 131, the anti-TF antibody affinity columns bind to TGFRt15-21s137L, which contains TF as a fusion partner for TGFRt15-21s 137L. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Example 58: production and characterization of TGFRt15-TGFRs21 fusion proteins

A fusion protein complex was generated comprising tgfβ receptor II/IL-15rαsu/IL-21 and tgfβ receptor II/TF/IL-15 fusion proteins (figures 132 and 133). Human TGF-beta receptor II (Ile 24-Asp 159), tissue factor 219, IL-21 and IL-15 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were prepared by ligating two TGF-beta receptor II sequences with a G4S (3) linker to produce a single-stranded form of TGF-beta receptor II, and then ligating directly to the N-terminal coding region of tissue factor 219, followed by ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of constructs comprising two TGF-beta receptor II linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRt15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the TGFRt15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly linking two tgfβ receptor II to the IL-15rαsu chain, followed by the N-terminal coding region of IL-21 synthesized by Jin Weizhi. The nucleic acid and protein sequences of constructs comprising TGF-beta receptor II linked to the N-terminus of IL-15RαSu followed by the N-terminus of IL-21 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRs21 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence (including the leader sequence) of the TGFRs21 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

TGFR/IL-15RαSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors as described previously (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al, transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72) and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble TGFR/TF/IL-15:TGFR/IL-15RαSu/IL-21 protein complexes (called TGFRt15-TGFRs 21), which can be purified by anti-TF antibody IgG1 affinity and other chromatographic methods.

Purification elution chromatography of TGFRt15-TGFRs21 using anti-TF antibody affinity column

TGFRt15-TGFRs21 harvested from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized samples were then buffer exchanged in PBS using an Amicon centrifuge filter with a molecular weight cutoff of 30 KDa. As shown in fig. 134, the anti-TF antibody affinity column binds to TGFRt15-TGFRs21 containing TF as a fusion partner. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Reduced SDS-PAGE analysis of TGFRt15-TGFRs21

To determine the purity and molecular weight of the protein, TGFRt15-TGFRs21 protein samples purified with anti-TF antibody affinity columns were analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) under reducing conditions. After electrophoresis, the gel was stained with instant blue for about 30min, and then decolorized in purified water overnight.

To verify that the TGFRt15-TGFRs21 protein undergoes glycosylation post-translationally in CHO cells, deglycosylation experiments were performed using the protein deglycosylation mixture II kit from the new england biology laboratory and the manufacturer's instructions. FIG. 135 shows the reduced SDS-PAGE analysis of samples in the non-deglycosylated (red outline lane 1) and deglycosylated (yellow outline lane 2) states. It is clear that the protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified samples showed the expected molecular weights (69 kDa and 55 kDa) in a reduced SDS gel. Lane M contains 10ul SeeBlue Plus2 pre-staining standard.

Immunostimulation of TGFRt15-TGFRs21 in C57BL/6 mice

TGFRt15-TGFRs21 is a multi-chain polypeptide (type a multi-chain polypeptide described herein) comprising a first polypeptide that is a soluble fusion of two tgfbrii domains, human tissue factor 219 fragment and human IL-15 (TGFRt 15) that is single chain and a second polypeptide that is a soluble fusion of two tgfbrii domains, sushi domain of human IL-15 receptor alpha chain and human IL-21 (TGFRs 21).

CHO cells were co-transfected with TGFRt15 and TGFRs21 vectors. The TGFRt15-TGFRs21 complex was purified from transfected CHO cell culture supernatant. As shown in FIG. 136, TGF-beta receptor, IL-15, IL-21 and Tissue Factor (TF) components in the complex were demonstrated by ELISA. TF in TGFRt15-TGFRs21 was determined using humanized anti-TF monoclonal antibodies (anti-TF IgG 1) as capture antibodies, and IL-15, TGF beta receptor and IL-21 in TGFRt15-TGFRs21 were determined using biotinylated anti-human IL-15 antibodies (Andi organism), biotinylated anti-human TGF beta receptor antibodies (Andi organism) and biotinylated anti-human IL-21 antibodies (Andi organism) as detection antibodies, respectively. For detection, peroxidase-conjugated streptavidin (jackson immunoresearch laboratory) and ABTS were used.

Wild type C57BL/6 mice were treated subcutaneously with control solution (PBS) or 3mg/kg of TGFRt15-TGFRs 21. Four days after treatment, spleen weight and the percentage of various immune cell types present in the spleen were assessed. As shown in fig. 137A, CD4 present in spleens of control-treated and TGFRt15-TGFRs 21-treated mice was assessed ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells. Dynamic proliferation of immune cells based on Ki67 expression following TGFRt15-TGFRs21 treatment was also assessed. Spleen cells were stained with fluorochrome-conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 (NK) antibodies and then stained intra-cell with anti-Ki 67 antibody. By passing throughFlow cytometry analysis of CD4 ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells, mean Fluorescence Intensity (MFI) of Ki67 for the corresponding immune cell subpopulation (fig. 137A and 137B). In addition, the cytotoxic potential of splenocytes induced by TGFRt15-TGFRs21 based on granzyme B expression after single dose treatment of mice was also assessed. As shown in FIG. 138, granzyme B expression of NK cells increased after treatment in spleen of mice treated with TGFRt15-TGFRs 21. Spleen cells from TGFRt15-TGFRs21 treated mice were stained with fluorochrome-conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 (NK) antibodies and then stained intra-cell with anti-granzyme B antibodies. The average fluorescence intensity (MFI) of granzyme B corresponding to the immune cell subpopulation was analyzed by flow cytometry (fig. 138).

As shown in FIG. 137A, CD8 on day 4 after single TGFRt15-TGFRs21 treatment in the spleen of mice treated with TGFRt15-TGFRs21 ⁺ The percentage of both T cells and NK cells increases. These results indicate that TGFRt15-TGFRs21 is capable of inducing immune cells to proliferate in the spleen of mice, in particular CD8 ⁺ T cells and NK cells.

In addition, mouse spleen cells were also evaluated for cytotoxicity to tumor cells. Mouse Yac-1 cells were labeled with CellTrace violet and used as tumor target cells. Spleen cells were prepared from TGFRt15-TGFRs 21-treated mice and used as effector cells. Target cells were mixed with effector cells in RPMI-10 medium with or without 100nM of TGFRt15-TGFRs21 at E: T ratio = 10:1 and incubated for 24 hours at 37 ℃. Target Yac-1 cell inhibition was assessed by analyzing ionomarked living Yac-1 cells using flow cytometry. The percentage of Yac-1 inhibition was calculated using the following formula: (1- [ Yac-1 viable cell count in the test sample ]/[ Yac-1 viable cell count in the spleen cell-free sample ]) x100. As shown in FIG. 139, splenocytes from mice treated with TGFRt15-TGFRs21 were more cytotoxic to Yac-1 cells than control mice cells in the presence of TGFRt15-TGFRs21 during cytotoxicity assays (FIG. 139).

Example 59: production of TGFRt15-TGFRs16 fusion proteins

A fusion protein complex was generated comprising tgfβ receptor II/IL-15rαsu/anti-CD 16scFv and tgfβ receptor II/TF/IL-15 fusion protein (fig. 140 and 141). Human TGF-beta receptor II (Ile 24-Asp 159), tissue factor 219 and IL-15 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were prepared by ligating two TGF-beta receptor II sequences with a G4S (3) linker to produce a single-stranded form of TGF-beta receptor II, and then ligating directly to the N-terminal coding region of tissue factor 219, followed by ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of constructs comprising two TGF-beta receptor II linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRt15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the TGFRt15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly linking two tgfβ receptor II to the IL-15rαsu chain, followed by an anti-CD 16scFv sequence synthesized by Jin Weizhi. The nucleic acid and protein sequences of constructs comprising TGF-beta receptor II linked to the N-terminus of IL-15RαSu followed by an anti-CD 16scFv sequence are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRs16 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

(anti-human CD16 scFv)

TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

The amino acid sequence (including the leader sequence) of the TGFRs16 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

(anti-human CD16 scFv)

SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

TGFR/IL-15RαSu/anti-CD 16scFv and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors as described previously (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al, transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.hum Gene Ther 2005; 16:457-72) and the expression vectors were transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble TGFR/TF/IL-15:TGFR/IL-15RαSu/anti-CD 16scFv protein complex (called TGFRt15-TGFRs 16) which is purified by anti-TF IgG1 affinity and other chromatographic methods.

Example 60: production of TGFRt15-TGFRs137L fusion protein

A fusion protein complex was generated comprising tgfβ receptor II/IL-15rαsu/CD137L and tgfβ receptor II/TF/IL-15 fusion proteins (fig. 142 and 143). Human TGF-beta receptor II (Ile 24-Asp 159), tissue factor 219, CD137L and IL-15 sequences were obtained from the UniProt website and the DNA of these sequences was synthesized by Jin Weizhi. Specifically, constructs were prepared by ligating two TGF-beta receptor II sequences with a G4S (3) linker to produce a single-stranded form of TGF-beta receptor II, and then ligating directly to the N-terminal coding region of tissue factor 219, followed by ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of constructs comprising two TGF-beta receptor II linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRt15 construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the TGFRt15 fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly linking two tgfβ receptor II to the IL-15rαsu chain, followed by a (G4S) 3 linker and a CD137L sequence synthesized by Jin Weizhi. The nucleic acid and protein sequences of constructs comprising TGF-beta receptor II linked to the N-terminus of IL-15RαSu followed by a (G4S) 3 linker and a CD137L sequence are shown below.

The nucleic acid sequence (including the signal peptide sequence) of the TGFRs137L construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human TGF-beta receptor II fragments)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S) 3 Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human CD 137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including the leader sequence) of the TGFRs137L fusion protein is as follows:

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human TGF-beta receptor II)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

((G4S) 3 Joint)

GGGGSGGGGSGGGGS

(human CD 137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

In some cases, the leader peptide is cleaved from the intact polypeptide to produce a mature form that may be soluble or secreted.

TGFR/IL-15RαSu/CD137L and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, yu Y, dudley ME, zheng Z, robbins PF, li Y et al, transfer of a TCR Gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005; 16:457-72), and the expression vectors transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble TGFR/TF/IL-15:TGFR/IL-15RαSu/CD137L protein complexes (called TGFRt15-TGFRs 137L), which can be purified by anti-TF IgG1 affinity and other chromatographic methods.

Example 61 production and characterization of exemplary Single-chain chimeric polypeptide 2t2

An exemplary single chain chimeric polypeptide is produced that includes a first target binding domain that binds to an IL-2 receptor, a soluble human tissue factor domain, and a second target binding domain (IL-2/TF/IL-2; referred to as 2t 2) that binds to an IL-2 receptor (FIG. 144). The nucleic acid and amino acid sequences of such single-chain chimeric polypeptides are shown below.

Nucleic acid encoding an exemplary Single-chain chimeric polypeptide (IL-2/TF/IL-2) (SEQ ID NO: 164)

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(first IL-2 fragment)

GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACC

(human tissue factor 219 form)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(second IL-2 fragment)

GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT

Exemplary Single chain chimeric polypeptide (IL-2/TF/IL-2) (SEQ ID NO: 163)

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-2)

aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistlt

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-2)

aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistlt

Nucleic acids encoding IL-2/TF/IL-2 were cloned into modified retroviral expression vectors as previously described (Hughes et al, hum Gene Ther 16:457-72, 2005). An expression vector encoding IL-2/TF/IL-2 was transfected into CHO-K1 cells. Expression of the expression vector in CHO-K1 cells allows secretion of the soluble IL-2/TF/IL-2 single chain chimeric polypeptide (referred to as 2t 2) which can be purified by anti-TF antibody affinity chromatography and other chromatographic methods.

IL-2 and 2t2 similarly promote proliferation of 32D beta cells containing IL-2R beta and common gamma chain

To assess IL-2 activity of 2t2, 2t2 was compared to recombinant IL-2 in promoting proliferation of 32D beta cells expressing IL-2Rbeta and a common gamma chain. IL-2 dependent 32D beta cells were washed 5 times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells were seeded into the wells. Serial dilutions of 2t2 or IL-2 were added to cells (figure 145). Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By adding 10. Mu.l WST1 to each well on day 3 and at CO ₂ Cell proliferation was detected by further incubation for 3 hours at 37℃in the incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in figure 145, 2t2 and IL-2 activated 32D beta cells in a similar manner. EC of 2t2 and IL-2 ₅₀ 158.1pM and 140pM, respectively.

2t2 shows an improved ability to promote proliferation of CTLL-2 cells containing IL-2Rαβγ compared to IL-2

To assess IL-2 activity of 2t2, 2t2 was compared to recombinant IL-2 in promoting proliferation of CTLL-2 cells expressing IL-2Rα, IL-2Rβ and a common gamma chain. IL-2 dependent CTLL-2 cells were washed 5 times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells were seeded into the wells. Serial dilutions of 2t2 or IL-2 were added to cells (figure 146). Cell in CO ₂ Incubation in incubator at 37℃for 3 days. By adding 10. Mu.l WST1 to each well on day 3 and at CO ₂ Cell proliferation was detected by further incubation for 3 hours at 37℃in the incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in FIG. 146, 2t2 promotes CTLL-2 cell proliferation 4-5 times stronger than IL-2. EC of 2t2 ₅₀ 123.2pM and IL-2 548.2pM.

2t2 inhibition of ApoE ^-/- High fat induced hyperglycemia exacerbation in mice

To six week old female ApoE ^-/- Mice (jackson laboratory) were fed standard feed (chow diet) or high fat feed containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein and 15% starch (TD 88137, halms laboratory (Harlan Laboratories)) and maintained under standard conditions. At week 7, high fat diet fed mice were randomized into control and treatment groups. Mice were then given a 3mg/kg dose of either 2t2 (treatment group) or PBS (feed group and control group) by subcutaneous injection. Three days after dosing, mice were fasted overnight and blood samples were collected by retroorbital venous plexus puncture. The overnight fasting blood glucose level was measured using an OneTouch glucometer. As shown in FIG. 147, the results indicate that 2t2 injection effectively inhibited ApoE ^-/- The glucose level of the mice increased.

2t2 CD4 in blood lymphocytes ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory (Treg) cell rate was significantly up-regulated

To six week old female ApoE ^-/- Mice (jackson laboratory) were fed standard feed or high fat feed containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein and 15% starch (TD 88137, halland laboratory) and maintained under standard conditions. At week 7, high fat diet fed mice were randomized into control and treatment groups. Mice were then given a 3mg/kg dose of either 2t2 (treatment group) or PBS (feed group and control group) by subcutaneous injection. On the third day after dosing, overnight fasting blood samples were collected by retroorbital venous plexus puncture and incubated with ACK lysis buffer (sammeler technique) for 5 minutes at 37 ℃. The samples were then resuspended in FACS buffer (1 XPBS (sea clone) containing 0.5% BSA (EMD Milibo)) And 0.001% sodium azide (sigma)) and surface stained with FITC-anti-CD 4 and APC-anti-CD 25 antibodies (hundred-in biotechnology) for 30 minutes. The surface stained samples were further fixed and pre-metallized with Fix/Perm buffer (hundred-in biotechnology) and stained intracellularly with PE-anti-Foxp 3 antibody (hundred-in biotechnology). After staining, cells were washed twice with FACS buffer, and then centrifuged at 1500RPM for 5 minutes at room temperature. Cells were analyzed by flow cytometry (Celesta-BD biosciences). As shown in FIG. 148, 2t2 treatment significantly increased the Treg population (3.5% + -0.32) in blood lymphocytes compared to the untreated group (0.4% + -0.16 for the feed group and 0.46% + -0.09 for the high fat feed group).

Purification elution chromatography of 2t2 from anti-TF antibody affinity column

2t2 collected from cell cultures was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample buffer was then exchanged into PBS using an Amicon centrifuge filter with a molecular weight cut-off of 30 kDa. As shown in fig. 149, the anti-TF antibody affinity column binds to 2t2 containing TF as the fusion domain. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (ph 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

Analytical Size Exclusion Chromatography (SEC) analysis of 2t2

To analyze 2t2 using analytical Size Exclusion Chromatography (SEC), a Superdex200 Increase 10/300 GL gel filtration column (from the general electric healthcare group) was connected to the AKTA Avant system (from the general electric healthcare group). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7mL/min. Samples containing 2t2 in PBS were injected into a Superdex200 column using a capillary loop and analyzed by SEC. SEC chromatograms of the samples are shown in figure 150. SEC results indicated two protein peaks at 2t 2.

2t2 reduced SDS-PAGE

To determine the purity and molecular weight of the protein, 2t2 protein samples purified with anti-TF antibody affinity columns were analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) under reducing conditions. After electrophoresis, the gel was stained with instant blue for about 30min, and then decolorized in purified water overnight.

To verify that the 2t2 protein undergoes glycosylation post-translationally in CHO cells, deglycosylation experiments were performed using the protein deglycosylation mixture II kit from the new england biology laboratory according to the manufacturer's instructions. FIGS. 151A and 151B show reduced SDS-PAGE analysis of samples in the non-deglycosylated (red outline lane 1) and deglycosylated (yellow outline lane 2) state. The results indicate that the 2t2 protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified samples were run in a reduced SDS gel at the expected molecular weight (56 kDa). Lane M was loaded with 10. Mu.L of SeeBlue Plus2 pre-stain.

In vivo characterization of 2t2

2t2 was subcutaneously injected into C57BL/6 mice at different doses to determine the immunostimulatory activity of 2t2 in vivo. Mice were treated subcutaneously with control solutions (PBS) or 2t2 at 0.1, 0.4, 2 and 10 mg/kg. On day 3 post-treatment, the treated mice were euthanized. Spleens of mice were collected and weighed on day 3 post-treatment. Preparation of a single spleen cell suspension and preparation of spleen cells directed against CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells were stained (anti-CD 4, anti-CD 8 and anti-NK 1.1 antibodies conjugated with fluorochromes) and analyzed by flow cytometry. The results showed that 2t2 was effective in expanding spleen cells, especially at 0.1-10mg/kg based on spleen weight (FIG. 152A). CD8 at 2 and 10mg/kg ⁺ The percentage of T cells was higher than the control treated mice (figure 152B). At all doses tested, the percentage of NK cells was higher than the control treated mice (figure 152B).

IL-2 is known to up-regulate CD25 expression in immune cells. We therefore learn about CD4 in 2t2 treated mice ⁺ T cells, CD8 ⁺ CD25 expression by T cells and NK cells. C57BL/6 mice were treated subcutaneously with 2t2 as described in the preceding paragraph. Spleen cells were stained with fluorochrome-conjugated anti-CD 4, anti-CD 8, CD25 and NK1.1 monoclonal antibodies. The spleen cell subpopulations were analyzed for CD25 expression (MFI) by flow cytometry. As shown in figure 153, 2t2 significantly upregulated CD4 at the doses and time points tested ⁺ CD25 expression by T cells, but not CD8+ T cells or NK cells.

The pharmacokinetics of 2t2 in C57BL/6 mice were also studied. 2t2 was subcutaneously injected into C57BL/6 mice at 1 mg/kg. Blood was drawn from the tail vein of the mice at various time points as shown in fig. 154, and serum was prepared. The 2t2 concentration was determined by ELISA (capture: anti-tissue factor antibody; detection: biotinylated anti-human IL-2 antibody followed by SA-HRP and ABTS substrates). Half-life of 2t2 was 1.83 hours, calculated using PK Solutions 2.0 (Summit Research Services).

2t2 reduction of ApoE ^-/- High fat induced formation of atherosclerotic plaques in mice

To six week old female ApoE ^-/- Mice (jackson laboratory) were fed standard or high fat feeds (21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein and 15% starch) (TD 88137, halland laboratory) and maintained under standard conditions. At week 7, high Fat Diet (HFD) fed mice were randomized into control and treatment groups. Mice were then subcutaneously administered a 3mg/kg dose of either 2t2 (treatment group) or PBS (feed group and control group) weekly for 4 weeks. At week 12, all mice were euthanized by isoflurane. The aorta was collected, opened longitudinally and stained with sudan IV solution (0.5%) using a frontal procedure. The percent plaque area (red, as shown in fig. 155A) relative to total aortic area was then quantified using Image J software. Fig. 155A shows a representative view of the atherosclerotic plaques for each group. Fig. 155B shows the results of quantitative analysis of atherosclerotic plaques for each group. The plaque area percentage of the control group (HF feed) was higher than that of the treatment group (hfd+2t2) Much more: 10.28% versus 4.68%.

Inhibition of progression of type 2 diabetes by 2t2

For male BKS.Cg-Dock7 ^m +/+Lepr ^db The mice were fed standard feed and received drinking water ad libitum. At six weeks of age, mice were randomly divided into control and treatment groups. The treatment group received 2t2 at 3mg/kg every two weeks by subcutaneous injection, while the control group received vehicle (PBS) alone. The fasting blood glucose levels were measured overnight weekly using an OneTouch glucometer. The results show that 2t2 effectively inhibits BKS.Cg-Dock7 ^m +/+Lepr ^db Glucose levels in/J mice increased (FIG. 156).

2t2 significantly up-regulates CD4 in blood lymphocytes after the first injection ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cell ratio

For male BKS.Cg-Dock7 ^m +/+Lepr ^db J (db/db) (Jackson laboratory) mice were fed standard feed and received drinking water ad libitum. At six weeks of age, mice were randomly divided into control and treatment groups. The treatment group received 2t2 at 3mg/kg every two weeks by subcutaneous injection, while the control group received vehicle (PBS) alone. Fourth day after the first drug injection, overnight fasting blood samples were collected and incubated with ACK lysis buffer (sammer feishier technology) for 5 minutes at 37 ℃. The samples were then resuspended in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) and surface stained with FITC-anti-CD 4 and APC-anti-CD 25 antibodies (hundred-biosciences) for 30 minutes. The surface stained samples were further fixed and pre-metallized with Fix/Perm buffer (hundred-in biotechnology) and stained intracellularly with PE-anti-Foxp 3 antibody (hundred-in biotechnology). After staining, the cells were washed twice with FACS buffer and analyzed by flow cytometry (Celesta-BD bioscience). Measurement of CD4 in blood lymphocytes ⁺ CD25 ⁺ FoxP3 ⁺ Tregs percentage. As shown in fig. 157, the results demonstrate that 2t2 significantly up-regulates the Tregs ratio in blood lymphocytes (×p)<0.05)。

Example 62 production and characterization of exemplary Single-chain chimeric polypeptide 15t15

A second exemplary single chain chimeric polypeptide is produced that includes a first target binding domain that binds to an IL-15 receptor, a soluble human tissue factor domain, and a second target binding domain (IL-15/TF/IL-15; referred to as 15t 15) that binds to an IL-15 receptor (FIG. 158). The nucleic acid and amino acid sequences of such single-chain chimeric polypeptides are shown below.

Nucleic acid encoding an exemplary Single-chain chimeric polypeptide (IL-15/TF/IL-15) (SEQ ID NO: 170)

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(first IL-15 fragment)

AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGC

(human tissue factor 219 form)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(second IL-15 fragment)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

Exemplary Single chain chimeric polypeptide (IL-15/TF/IL-15) (SEQ ID NO: 169)

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid encoding IL-15/TF/IL-15 was cloned into a modified retroviral expression vector as previously described (Hughes et al, hum Gene Ther 16:457-72, 2005). The expression vector encoding IL-15/TF/IL-15 was transfected into CHO-K1 cells. Expression of the expression vector in CHO-K1 cells allows secretion of a soluble IL-15/TF/IL-15 single chain chimeric polypeptide (designated 15t 15), which can be purified by affinity to anti-TF antibodies and other chromatographic methods.

15t15 promotes proliferation of 32D beta cells containing IL-2R beta and a common gamma chain

IL-15 activity on 15t15 was compared to recombinant IL-15 in 32D beta cells expressing IL2R beta and a common gamma chain. IL-15 dependent 32D beta cells were washed five times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells were seeded into the wells. Serial dilutions of 15t15 or IL-15 were added to cells (fig. 159). Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By adding 10. Mu.l WST1 to each well on day 3 and at CO ₂ In the incubator at 37 DEG CCell proliferation was detected by culturing for 3 hours. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in fig. 159, 15t15 promoted 32D beta cell proliferation less effectively than IL-15. EC of 15t15 and IL-15 ₅₀ 161.4pM and 1.6pM, respectively.

Purification elution chromatography from 15t15 of anti-TF antibody affinity column

15t15 collected from cell cultures was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS followed by 6 column volumes of 0.1M acetic acid (pH 2.9). The A280 elution peaks were collected and then neutralized to pH 7.5-8.0 with 1M Tris base. The neutralized sample buffer was then exchanged into PBS using an Amicon centrifuge filter with a molecular weight cut-off of 30 kDa. As shown in figure 160, the anti-TF antibody affinity column binds to 15t15 containing TF as the fusion domain. The buffer exchanged protein samples were stored at 2-8 ℃ for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1M glycine (pH 2.5). The column was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was attached to the universal electric medical community AKTA Avant system. The flow rate of all steps except the elution step was 4mL/min, and the flow rate of the elution step was 2mL/min.

15t15 reduced SDS-PAGE

To determine the purity and molecular weight of the protein, 15t15 protein samples purified with anti-TF antibody affinity columns were analyzed by sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) under reducing conditions. After electrophoresis, the gel was stained with instant blue for about 30min, and then decolorized in purified water overnight.

To verify that 15t15 protein underwent glycosylation post-translationally in CHO cells, deglycosylation experiments were performed using protein deglycosylation mixture II kit from new england biology laboratories and manufacturer's instructions. FIGS. 161A and 161B show reduced SDS-PAGE analysis of samples in the non-deglycosylated (red outline lane 1) and deglycosylated (yellow outline lane 2) states. The results indicate that the 15t15 protein is glycosylated when expressed in CHO cells. After deglycosylation, the purified samples were run in a reduced SDS gel at the expected molecular weight (50 kDa). Lane M was loaded with 10. Mu.L of SeeBlue Plus2 pre-stain.

Example 63: stimulation of NK cells in vitro

A set of experiments was performed to assess the change in surface phenotype of NK cells after stimulation with 18t15-12s, 18t15-12s16 and 7t15-21s+ anti-TF antibodies. In these experiments, fresh human leukocytes were obtained from a blood bank and CD56 was isolated using Rosetteep/human NK cell reagent (Stem cell technology) ⁺ NK cells. Purity of NK cells>90% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE and CD69-APCFire750 antibodies (hundred-Biotech). Cells were counted and at 0.2x10 ⁶ The concentration of/mL was resuspended in 0.2mL of complete medium (RPMI 1640 (Ji Bike) supplemented with 2mM L-glutamine (sammer life technology), penicillin (sammer life technology), streptomycin (sammer life technology) and 10% fbs (sea clone)) in flat bottom 96-well plates. At 37 ℃ and 5% CO ₂ The cells were then stimulated for 16 hours with the following substances: 18t15-12s (100 nM); 18t15-12s16 (100 nM); a mixture of the single cytokines rhIL15 (50 ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (invitrogen) and rhIL-12 (10 ng/mL) (peprotec); 7t15-21s+ anti-TF antibody (100 nM-50 nM); 7t15-21s (100 nM); or an anti-TF antibody (50 nM). The next day, cells were harvested and surface stained with CD56, CD16, CD25, CD69, CD27, CD62L, NKp and NKp44 specific antibodies for 30 minutes. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After two washes, the cells were analyzed by flow cytometry (Celesta-BD biosciences). FIGS. 162A and 162B show that incubation of purified NK cells with 18t15-12s, 18t15-12s16 and 7t15-21s+ anti-TF antibodies overnight increased the percentage of cells expressing CD25, CD69, NKp44 and NKp30 activation markers and decreased the percentage of cells expressing CD 62L. All activation marker data were derived from CD56 ⁺ A portal lymphocyte.

A set of experiments was performed to assess the change in surface phenotype of lymphocyte populations after stimulation with 18t15-12s, 18t15-12s16 and 7t15-21 s. In these experiments, fresh human leukocytes were obtained from a blood bank. Peripheral blood lymphocytes were isolated using Ficoll-PAQUE Plus (general electric medical group) density gradient medium. Cells were counted and at 0.2x10 ⁶ The concentration of/mL was resuspended in 0.2mL of complete medium (RPMI 1640 (Ji Bike) supplemented with 2mM L-glutamine (sammer life technology), penicillin (sammer life technology), streptomycin (sammer life technology) and 10% fbs (sea clone)) in flat bottom 96-well plates. At 37℃and 5% CO ₂ The cells were stimulated for 16 hours with the following: a mixture of 18t15-12s (100 nM), 18t15-12s16 (100 nM), the single cytokine rhIL-15 (50 ng/mL) (Miltian, miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12 (10 ng/mL) (Peprotech); 7t15-21s (100 nM) +anti-TF antibody (50 nM); 7t15-21s (100 nM); or an anti-TF antibody (50 nM). The following day, cells were harvested and surface stained with CD4 or CD8, CD62L and CD69 specific antibodies for 30 minutes. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone) containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma) (5 min at 1500 RPM) after two washes, the cells were analyzed by flow cytometry (Celesta-BD biosciences). Figure 163 shows purified lymphocyte populations (CD 4 and CD8T cells) were incubated overnight with 18T15-12s, 18T15-12s16 or 7T15-21s+ anti-TF antibodies such that the percentage of CD8 and CD 4T cells expressing CD69 was increased. Additionally, incubation with 7T15-21s+ anti-TF antibodies such that the percentage of CD8 and CD 4T cells expressing CD62L was increased (figure 163).

A set of experiments was performed to determine the effect of 18t15-12s on the extracellular acidification rate (ECAR) of NK cells purified from human blood. ECAR can be used to measure glycolysis. Glycolysis is the chemical conversion of glucose in one molecule into pyruvate in two molecules in a cell, producing ATP in two molecules simultaneously. An increase in ECAR measured by the Seahorse XF96 analyzer indicates an increase in glycolysis. In these experiments, fresh human leukocytes were obtained from a blood bank and used with RosetteSeIsolation of CD56 by p/human NK cell reagent (Stem cell technology) ⁺ N cells. Purity of NK cells>70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE and CD69-APCFire750 antibodies (hundred-Biotech). Cells were counted and at 0.2x10 ⁶ The concentration of/mL was resuspended in 0.2mL of complete medium (RPMI 1640 (Ji Bike) supplemented with 2mM L-glutamine (sammer life technology), penicillin (sammer life technology), streptomycin (sammer life technology) and 10% fbs (sea clone)) in flat bottom 96-well plates. The DEG C cells are at 37 ℃ and 5% CO ₂ The single cytokines hIL-12 (10 ng/mL) (hundred-in Biotechnology), hIL-18 (50 ng/mL) (R&D) And hIL-15 (50 ng/mL) (NCI) or 18t15-12s (100 nM) for 14-18 hours. The next day, cells were harvested and washed twice in Seahorse medium. Cells (2X 10) ⁵ Individual cells/well) were inoculated into 96-well flux plates coated with 10 μl of poly-L-lysine (sigma). NK cells were allowed to adhere to the plate for 30 min prior to analysis. Glucose, oligomycin and 2DG solutions were prepared at 10-fold concentration in buffered Seahorse medium and injected into ports A, B and C of the calibration plate. ECAR readings were taken every 6.5-7 minutes, and ECAR results represent average readings over 80 minutes or average readings at each time point. FIG. 164 shows that 18t15-12s overnight stimulation of NK cells resulted in elevated basal ECAR levels. The addition of glucose and oligomycin further showed glycolysis and glycolytic capacity enhancement, respectively, of NK cells stimulated overnight with 18t15-12s (figure 164). NK cells treated overnight with either medium alone or a mixture of IL12, IL18 and IL-15 were used for comparison (FIG. 164).

A set of experiments was performed to determine the increase in levels of phosphorylated STAT4 and phosphorylated STAT5 in NK cells after stimulation with 18t15-12 s. In these experiments, fresh human leukocytes were obtained from a blood bank and CD56 was isolated using Rosetteep/human NK cell reagent (Stem cell technology) ⁺ N cells. Purity of NK cells>70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE and CD69-APCFire750 specific antibodies (hundred-Biotech). Cells were counted and at 0.05x10 ⁶ A concentration of/mL was resuspended in 96-well flat bottom plate at 0.1mL complete medium (RPMI 1640 (Ji Bike), supplemented with 2mM L-trough)Ammonia amide (Sesammer life technology), penicillin (Sesammer life technology), streptomycin (Sesammer life technology) and 10% FBS (sea clone)). At 37 ℃ and 5% CO ₂ Cells were stimulated with hIL-12 (10 ng/mL) (hundred-in Biotechnology) or hIL-15 (50 ng/mL) (NCI) (single cytokine) or 18t15-12s (100 nM) for 90 min. Unstimulated NK cells (US) were used as controls. Cells were harvested and fixed in paraformaldehyde (sigma) to a final concentration of 1.6%. Plates were incubated in the dark at room temperature for 10 minutes. FACS buffer (1 XPBS (sea clone), containing 0.5% BSA (EMD Miibo) and 0.001% sodium azide (Sigma)) was added (100. Mu.L) and cells were transferred to 96 well "V" plates. Cells were washed at 1500RPM for 5 minutes at room temperature. The cell pellet was mixed with 100 μl of cooled methanol by gentle pipetting up and down, and the cells were incubated at4 ℃ for 30 min. The cells were mixed with 100mL FACS buffer and washed at 1500RPM for 5 minutes at room temperature. The cell pellet was mixed with 50mL FACS buffer containing 4mL pSTAT4 (BD Bioscience) and pSTAT5 antibody (BD Bioscience) followed by incubation in the dark for 30 minutes at room temperature. The cells were mixed with 100mL FACS buffer and washed at 1500RPM for 5 minutes at room temperature. The cell pellet was mixed with 50mL FACS buffer and the cells were analyzed by flow cytometry (Celesta-BD bioscience). FIG. 165 shows that NK cells incubated with 18t15-12s induced increases in pSTAT4 and pSTAT5 (data plotted, normalized fold change).

A set of experiments was performed to determine the effect of 18t15-12s or cytokine mixtures (e.g., IL12, IL18, and IL-15) on the Oxygen Consumption Rate (OCR) and extracellular acidification rate (ECAR) of NK cells purified from human blood. OCR and ECAR were measured by Seahorse XF96 analyzer. In these experiments, fresh human NK cells were isolated from human leukocytes via negative selection using RosetteSep/human NK cell reagent (stem cell technology). Freshly purified NK cells were stimulated overnight (16 hours) with 18t15-12s (100 nM) or a mixture of rhIL12 (10 ng/mL), rhIL18 (50 ng/mL) and rhIL-15 (50 ng/mL) cytokines as controls. The next day, cells were washed, counted, and equal numbers of cells were inoculated into buffered Seahorse medium. Glucose, oligomycin and 2DG solutions were prepared at 10-fold concentration in buffered Seahorse medium and injected into ports A, B and C of the calibration plate. Figure 166 shows OCR (left) and ECAR (right) data from two individual donors. Stimulation of NK cells overnight at 18t15-12s resulted in elevated basal ECAR and OCR levels. The addition of glucose and oligomycin further showed an increase in glycolysis and glycolytic capacity, respectively, of NK cells stimulated overnight with 18t15-12 s. NK cells treated overnight with either medium alone or a mixture of IL12, IL18 and IL-15 were used for comparison.

Example 64:2t2 and/or TGFRt15-TGFRs stimulate NK cells in vivo

A set of experiments was performed to determine the effect of the 2t2 construct on immune stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solution (PBS) or 2t2 at 0.1, 0.4, 2 and 10 mg/kg. The treated mice were euthanized 3 days after treatment. Spleen weights were measured and single spleen cell suspensions were prepared. Spleen cell suspensions were stained with conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 (NK) antibodies. Analysis of CD4 by flow cytometry ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells, and CD25 expression on lymphocyte subpopulations. FIG. 167A shows that 2t2 is effective to expand spleen cells, particularly at a dose level of 0.1-10mg/kg, based on spleen weight. After treatment, CD8 in 2t2 treated mice at 2 and 10mg/kg ⁺ The percentage of T cells was higher than the control treated mice (fig. 167B). The NK cell percentage was also higher in the 2t2 treated mice than in the control treated mice at all the doses tested at 2t2 (fig. 167B). In addition, 2t2 significantly upregulated CD4 after treatment at 0.4 to 10mg/kg ⁺ T cells other than CD8 ⁺ CD25 expression by T cells and NK cells (fig. 167C).

A set of experiments was performed to determine the effect of the TGFRt15-TGFRs construct on immunostimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solutions (PBS) or with TGFRt15-TGFRs of 0.3, 1, 3 and 10 mg/kg. 4 days after treatment, the treated mice were euthanized. Spleen weights were measured and single spleen cell suspensions were prepared. Spleen cell suspensions were stained with conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 (NK) antibodies. By passing throughFlow cytometry analysis of CD4 ⁺ T cells, CD8 ⁺ T cell and NK cell percentages. FIG. 168A shows that spleen weight of mice treated with TGFRt15-TGFRs increased with increasing dose of TGFRt 15-TGFRs. Furthermore, mice treated with 1mg/kg, 3mg/kg and 10mg/kg TGFRt15-TGFRs had higher spleen weights than control solution treated mice. FIG. 168B shows CD8 ⁺ The percentage of both T cells and NK cells increased with increasing TGFRt15-TGFRs dose. Specifically, CD8 in mice treated with 0.3mg/kg, 3mg/kg and 10mg/kg TGFRt15-TGFRs compared to control treated mice ⁺ The percentage of T cells was higher and the percentage of NK cells was higher in mice treated with 0.3mg/kg, 1mg/kg, 3mg/kg and 10mg/kg TGFRt15-TGFRs compared to control treated mice.

A set of experiments was performed to determine apoE of TGFRt15-TGFRs constructs or 2t2 constructs in Western fed diet ^-/- Immunostimulation was performed in mice. In these experiments, 6 week old females were given B6.129P2-ApoE ^tm1Unc Mice (jackson laboratory) were fed western diet (TD 88137, envigo Laboratories) containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein and 15% starch. After 8 weeks of western feed, 3mg/kg of TGFRt15-TGFRs or 2t2 were subcutaneously injected into mice. Three days after treatment, mice were fasted for 16 hours and then blood samples were collected by retroorbital venous plexus puncture. Blood was mixed with 10 μl of 0.5M EDTA and 20 μl of blood was drawn for lymphocyte subpopulation analysis. The red blood cells were treated with ACK (0.15M NH) ₄ Cl，1.0mM KHCO ₃ ，0.1mM Na ₂ EDTA, pH 7.4) and lymphocytes were stained with anti-mouse CD8a and anti-mouse NK1.1 antibody in FACS staining buffer (1% BSA in PBS) at 4℃for 30 min. Cells were washed once and analyzed with BD FACS Celesta. For Treg staining, ACK-treated blood lymphocytes were stained with anti-mouse CD4 and anti-mouse CD25 antibodies for 30 min at 4 ℃ in FACS staining buffer. Cells were washed once and resuspended in fixation/permeabilization working solution and incubated for 60 minutes at room temperature. Cells were washed once and resuspended in permeabilization buffer. The samples were centrifuged at 300-400x g for 5 minutes at room temperature, And then the supernatant was discarded. The cell pellet was resuspended in the remaining volume and the volume was adjusted to about 100 μl with 1x permeabilization buffer. anti-Foxp 3 antibodies were added to the cells and the cells were incubated at room temperature for 30 minutes. Permeabilization buffer (200 μl) was added to the cells and the cells were centrifuged at 300-400x g for 5 min at room temperature. Cells were resuspended in flow cytometry staining buffer and analyzed on a flow cytometer. FIGS. 169B-169C show that TGFRt15-TGFRs treatment resulted in ApoE fed Western diet ^-/- NK cells and CD8 in mice ⁺ The percentage of T cells increases. Figure 169A shows that 2t2 treatment also increased the percentage of Treg cells.

Example 65: induction of immune cell proliferation in vivo

A set of experiments was performed to determine the effect of the 2t2 construct in stimulating immune cells in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solution (PBS) or 2t2 at 0.1, 0.4, 2 and 10 mg/kg. The treated mice were euthanized 3 days after treatment. Spleen weights were measured and single spleen cell suspensions were prepared. Spleen cell suspensions were stained with conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 (NK) antibodies. Analysis of CD4 by flow cytometry ⁺ T cells, CD8 ⁺ Percentages of T cells and NK cells. FIG. 170A shows that 2t2 treatment is effective in expanding spleen cells, especially at 0.1-10mg/kg based on spleen weight. CD8 at 2 and 10mg/kg ⁺ The percentage of T cells was higher than the control treated mice (figure 170B). In addition, the percentage of NK cells was higher than control treated mice at all tested doses of 2t2 (fig. 170B). These results indicate that 2t2 treatment is capable of inducing CD8 in C57BL/6 mice ⁺ T cell and NK cell proliferation.

A set of experiments was performed to determine the effect of the TGFRt15-TGFRs construct on immunostimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solutions (PBS) or with TGFRt15-TGFRs of 0.1, 0.3, 1, 3 and 10 mg/kg. 4 days after treatment, the treated mice were euthanized. Spleen weight was measured and spleen cell suspensions were prepared. Spleen cell suspensions were treated with conjugated anti-CD 4, anti-CD 8 and anti-NK 1.1 (NK) antibody staining. Cells were additionally stained for the proliferation marker Ki 67. FIG. 171A shows that spleen weight of mice treated with TGFRt15-TGFRs increased with increasing dose of TGFRt 15-TGFRs. In addition, the spleen weights were higher in mice treated with 1mg/kg, 3mg/kg and 10mg/kg of TGFRt15-TGFRs than mice treated with control solution alone. CD8 ⁺ The percentage of both T cells and NK cells increased with increasing TGFRt15-TGFRs dose (fig. 171B). Finally, at all tested doses of TGFRt15-TGFRs, TGFRt15-TGFRs gave CD8 ⁺ Expression of the cell proliferation marker Ki67 in T cells and NK cells was significantly up-regulated. These results indicate that TGFRt15-TGFRs treatment induced CD8 in C57BL/6 mice ⁺ Proliferation of both T cells and NK cells.

A set of experiments was performed to determine apoE of TGFRt15-TGFRs constructs or 2t2 constructs in Western fed diet ^-/- Immunostimulation was performed in mice. In these experiments, 6 week old females were given B6.129P2-ApoE ^tm1Unc Mice (jackson laboratory) were fed western diet (TD 88137, envigo Laboratories) containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein and 15% starch. After 8 weeks of western feed, 3mg/kg of TGFRt15-TGFR or 2t2 was subcutaneously injected into mice. Three days after treatment, mice were fasted for 16 hours and then blood samples were collected by retroorbital venous plexus puncture. Blood was mixed with 10 μl of 0.5M EDTA and 20 μl of blood was drawn for lymphocyte subpopulation analysis. The red blood cells were treated with ACK (0.15M NH) ₄ Cl，1.0mM KHCO ₃ ，0.1mM Na ₂ EDTA, pH 7.4) and lymphocytes were stained with anti-mouse CD8a and anti-mouse NK1.1 antibody in FACS staining buffer (1% BSA in PBS) at 4℃for 30 min. Cells were washed once and resuspended in fixation buffer (bai biotech cat# 420801) for 20 min at room temperature. Cells were centrifuged at 350x g for 5 min, fixed cells were resuspended in intracellular dye-through wash buffer (hundred-Biotechnology Cat # 421002) and then centrifuged at 350x g for 5 min. Cells were then stained with anti-Ki 67 antibody for 20 min at RT. Cells were washed twice with intracellular stain permeabilization wash buffer and centrifuged at 350x g for 5 minutes. Then Cells were resuspended in FACS staining buffer. Lymphocyte subpopulations were analyzed with BD FACS Celesta. Treatment of ApoE with TGFRt15-TGFRs as depicted in FIG. 172A ^-/- Mouse induced NK and CD8 ⁺ T cell proliferation (Ki 67 positive staining). In addition, FIG. 172B shows that 2t2 treatment of ApoE ^-/- Mice also induced NK and CD8 ⁺ T cell proliferation (Ki 67 positive staining).

A set of experiments was performed to determine the effect of 7t15-21s+ anti-TF antibodies on NK cell expansion in NSG mice after 7t15-21s, TGFRt15-TGFRs and 2t2 treatments. In these experiments, fresh human leukocytes were obtained from a blood bank and CD56 was isolated using Rosetteep/human NK cell reagent (Stem cell technology) ⁺ NK cells. Purity of NK cells>90% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE and CD69-APCFire750 antibodies (hundred-Biotech). Cells were counted and at 2x10 ⁶ the/mL was resuspended in 2mL of complete medium (RPMI 1640 (Ji Bike), supplemented with 2mM L-glutamine (Sesammer life technologies), penicillin (Sesammer life technologies), streptomycin (Sesammer life technologies) and 10% FBS (sea clones)) in a 24 well flat bottom plate. Cells were stimulated for 15 days with the following substances: 7t15-21s (100 nM) and anti-TF antibody (50 nM). Every 2 days, cells were plated at 2X10 ⁶ the/mL was resuspended in fresh medium containing 100nM 7t15-21s and 50nM anti-TF antibody. As the culture volume increases, cells are transferred to a larger volume flask. Cells were counted using trypan blue to learn the fold expansion. NK cells amplified with 7t15-21s+ anti-TF antibody were washed three times in warm HBSS buffer (sea cloning) at 1000RPM at room temperature for 10 minutes each. NK cells amplified with 7t15-21s+ anti-TF antibody were resuspended in 10X10 ⁶ 0.2mL of HBSS buffer and was injected intravenously into the tail vein of NSG mice (NOD scid common gamma mice) (Jackson laboratories). Transferred NK cells were supported every 48 hours with 7t15-21s (10 ng/dose, i.p.), TGFRt15-TGFRs (10 ng/dose, i.p.), or 2t2 (10 ng/dose, i.p.) for up to 21 days. Implantation and persistence of human 7t15-21s+ anti-TF antibody-expanded NK cells, which were stained for hCD45, mCD45, hCD56, hCD3 and hCD16 antibodies, in blood was measured weekly by flow cytometry (Celesta-BD bioscience)(data represent 3 mice per group). FIG. 173 shows that treatment of mice with NK cells expanded with insemination transferred 7t15-21s+ anti-TF antibody induced NK cell expansion and persistence of insemination transferred with 7t15-21s, TGFRt15-TGFRs or 2t2 compared to control treated mice.

Example 66: NK-mediated cytotoxicity after treatment with single-or multiple-stranded constructs

A set of experiments was performed to determine whether treatment of NK cells with TGFRt15-TGFRs enhanced NK cell cytotoxicity. In these experiments, human Daudi (Daudi) B lymphoma cells were labeled with CellTrace violet (CTV) and used as tumor target cells. 4 days after 3mg/kg of TGFRt15-TGFRs subcutaneous treatment, the spleen of C57BL/6 female mice was isolated using a magnetic cell sorting method (Meitian-Ind Biotechnology (Miltenyi Biotec)) using NK1.1 positive selection. Human NK effector cells were isolated from peripheral blood mononuclear cells derived from human blood buffy coats (buffy coats) using Rosetteep/human NK cell reagent (Stem cell technology). Target cells (human ledi B lymphoma cells) were mixed with effector cells (mouse NK effector cells or human NK effector cells) in the presence of 50nm TGFRt15-TGFRs or in the absence of TGFRt15-TGFRs (control) and incubated at 37 ℃ for 44 hours for mouse NK cells and 20 hours for human NK cells. Target cell (daodi) viability was assessed by analyzing propidium iodide positive, CTV labeled cells using flow cytometry. The percentage of Daudi inhibition was calculated using the formula (1-number of viable tumor cells in experimental samples/number of viable tumor cells in samples without NK cells) x 100. Fig. 174 shows that after NK cells were activated with TGFRt15-TGFRs, the mouse (fig. 174A) and human (fig. 174B) NK cells had significantly stronger cytotoxicity to daodib cells than in the absence of TGFRt15-TGFRs activation.

A set of experiments was performed to determine Antibody Dependent Cellular Cytotoxicity (ADCC) of mice and human NK cells after treatment with TGFRt 15-TGFRs. In these experiments, human dado B lymphoma cells were labeled with CellTrace violet (CTV) and used as tumor target cells. 4 days after 3mg/kg of TGFRt15-TGFRs subcutaneous treatment, the spleen of C57BL/6 female mice was isolated with NK1.1 positive selection using a magnetic cell sorting method (Meitianfu Biotechnology). Human NK effector cells were isolated from peripheral blood mononuclear cells derived from human blood buffy coats (buffy coats) using Rosetteep/human NK cell reagent (Stem cell technology). Target cells (daodib cells) were mixed with effector cells (mouse NK effector cells or human NK effector cells) in the presence of anti-CD 20 antibody (10 nM Rituximab, genetec (Genentech)) and 50nM TGFRt15-TGFRs or no TGFRt15-TGFRs (control) and incubated for 44 hours at 37 ℃ for mouse NK cells and 20 hours for human NK cells. Daodib cells express CD20 targets of anti-CD 20 antibodies. Target cell viability was assessed by analyzing propidium iodide-positive, CTV-labeled target cells using flow cytometry after incubation. The percentage of Daudi inhibition was calculated using the formula (1-number of viable tumor cells in experimental samples/number of viable tumor cells in samples without NK cells) x 100. FIG. 175 shows that after NK cells were activated with TGFRt15-TGFRs, mouse NK cells (FIG. 175A) and human NK cells (FIG. 175B) had stronger ADCC activity against daodib cells than in the absence of TGFRt15-TGFRs activation.

A set of experiments was performed to determine the cytotoxicity of TGFRt15-TGFRs activated mouse NK cells on senescent B16F10 melanoma cells. In these experiments, mouse NK cells were activated in vivo by injecting 10mg/kg of TGFRt15-TGFRs into C57BL/6 mice for 4 days, followed by isolation of spleen NK cells. NK cells were then expanded in vitro in the presence of 100nM 2t2 for 7 days. Senescent B16F10 target cells (B16F 10-SNC) were labeled with CellTrace violet (CTV) and incubated with activated mouse NK effector cells at different effector to target (E: T) ratios for 16 hours. Cells were digested with trypsin, washed and resuspended in complete medium containing Propidium Iodide (PI) solution. The cytotoxicity of TGFRt15-TGFRs/2t2 activated NK cells against senescent cells was known by flow cytometry based on PI staining of CTV-labeled cells. These findings indicate that activation of NK cells with TGFRt15-TGFRs in vivo followed by expansion and activation with 2t2 in vitro resulted in increased killing of senescent melanoma tumor cells by NK cells (figure 176).

Example 67: treatment of cancer, diabetes and atherosclerosis

A set of experiments was performed to evaluate the anti-tumor activity of TGFRt15-TGFRs plus anti-TRP 1 antibody (TA 99) in combination chemotherapy in a melanoma mouse model. In these experiments, 0.5x10 will be ⁶ Individual B16F10 melanoma cells were subcutaneously injected into C57BL/6 mice. Mice were treated with three doses of docetaxel chemotherapy (10 mg/kg) (DTX) on days 1, 4 and 7 followed by a single dose of TGFRt15-TGFRs (3 mg/kg) +anti-TRP 1 antibody TA99 (200 μg) on day 9. FIG. 177A shows a schematic diagram of a processing scheme. Tumor growth was monitored by caliper measurement and the formula v= (l×w was used ² ) Tumor volume was calculated by/2, where L is the maximum tumor diameter and W is the vertical tumor diameter. Fig. 117B shows that treatment with dtx+tgfrt15-tgfrs+ta99 significantly reduced tumor growth (n=10,/p) compared to saline control and DTX treated groups<0.001, multiplex t-test analysis).

To assess immune cell subpopulations in the B16F10 tumor model, peripheral blood analysis was performed. In these experiments, C57BL/6 mice were injected with B16F10 cells and treated with DTX, DTX+TGFRt15-TGFRs+TA99 or saline. The submaxillary vein of B16F10 tumor bearing mice was sampled for day 2, day 5, day 8, and for day 11 after tumor injection in dtx+tgfrt15-tgfrs+ta99 groups of immunotherapy. RBCs were lysed in ACK lysis buffer and lymphocytes were washed and stained with anti-NK 1.1, anti-CD 8 and anti-CD 4 antibodies. Cells were analyzed by flow cytometry (Celesta-BD biosciences). FIGS. 177C-177E show that DTX+TGFRt15-TGFRs+TA99 treatment induces NK cells and CD8 in tumors compared to saline and DTX treated groups ⁺ The percentage of T cells increases.

On day 17, trizol was used to extract total RNA from tumors of mice treated with saline, DTX or DTX+TGFRt15-TGFRs+TA99. Total RNA (1. Mu.g) was used for cDNA synthesis using the QuantiTect reverse transcription kit (Kaiji). Primers designed in advance using FAM-labeled senescent cell markers (F) p21 (G) DPP4 and (H) IL6 were used with CFX96 detection system (burle (Bio-Rad)) for real-time PCR. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, expression of each target mRNA was based on Ct, according to 2 ^-Δ(ΔCt) Calculation, wherein Δct=ct _Target(s) -Ct _18S . The data are presented as fold change over saline control. FIGS. 177F-177H show that DTX treatment induced an increase in senescent tumor cells, which were subsequently decreased after treatment with TGFRt15-TGFRs+TA99 immunotherapy.

A set of experiments was performed to study 2t2 for Western feed induced ApoE ^-/- Improvement of hyperglycemia in mice. In these experiments, 6 week old females were given B6.129P2-ApoE ^tm1Unc Mice (jackson laboratory) were fed western diet (TD 88137, envigo Laboratories) containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein and 15% starch. After 8 weeks of western feed, 3mg/kg of TGFRt15-TGFRs or 2t2 were subcutaneously injected into mice. Three days after treatment, mice were fasted for 16 hours and then blood samples were collected by retroorbital venous plexus puncture. Blood glucose was measured with a blood glucose meter (OneTouch UltraMini) and a genuirimated test strip using a drop of fresh blood. As shown in fig. 178A, TGFRt15-TGFRs treatment significantly reduced western feed-induced hyperglycemia (p <0.04). Plasma insulin and resistin levels were analyzed using Eve Technologies' mouse rat metabolic array (Mouse Rat Metabolic Array). HOMA-IR was calculated using the following formula: steady state model evaluation-insulin resistance = blood glucose (mg/dL) × insulin (mU/mL)/405. As shown in figure 178B, both 2t2 and TGFRt15-TGFRs treatments reduced insulin resistance compared to the untreated group. As shown in fig. 178C, 2t2 (p<0.02 With TGFRt15-TGFRs (p)<0.05 Any significant decrease in resistin levels, which may be associated with a decrease in insulin resistance induced by 2t2 and TGFRt15-TGFRs (fig. 3B).

Example 68: memory-like NK cells induced by induction of NK cell differentiation into cytokine

A set of experiments was performed to evaluate NK cell differentiation into cytokine-induced memory-like NK cells (CIMK-NK cells) after stimulation with 18t15-12 s. In these experiments, the blood pool was used as the blood sourceFresh human white blood cells were obtained and CD56 was isolated using Rosetteep/human NK cell reagent (Stem cell technology) ⁺ NK cells. Purity of NK cells>90% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE and CD69-APCFire750 antibodies (hundred-Biotech). Cells were counted and at 2x10 ⁶ the/mL was resuspended in 2mL of complete medium (RPMI 1640 (Ji Bike), supplemented with 2mM L-glutamine (Sesammer life technologies), penicillin (Sesammer life technologies), streptomycin (Sesammer life technologies) and 10% FBS (sea clones)) in a 24 well flat bottom plate. No stimulation of cells ("no addition") or use of 18t15-12s (100 nM) or a single cytokine mixture ("single cytokine") comprising rhIL15 (50 ng/mL) (meitian-zender), rhIL18 (50 ng/mL) (Inweijie) and rhIL-12 (10 ng/mL) (Pepritec) at 37℃and 5% CO ₂ Lower stimulation for 16 hours. The next day, cells were harvested and washed twice with warm complete medium at 1000RPM for 10 minutes at room temperature. The cells were treated at 2X10 ⁶ the/mL was resuspended in 2mL of complete medium containing rhIL-15 (1 ng/mL) in a flat bottom 24-well plate. After every 2 days, half of the medium was replaced with fresh complete medium containing rhIL-15.

To assess the change in NK cell memory phenotype on day 7, cells were stained with antibodies to CD56, CD16, CD27, CD62L, NKp and NKp44 (hundred-in biotechnology) on the cell surface. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After two washes, the cells were analyzed by flow cytometry (Celesta-BD biosciences). FIG. 179 shows that NK cells were incubated with 18t15-12s such that CD16 expressing CD27, CD62L and NKp44 ⁺ CD56 ⁺ Increased percentage of NK cells and CD16 ⁺ CD56 ⁺ NKp30 levels (MFI) in NK cells are increased.

EXAMPLE 69 upregulation of CD44 memory T cells

C57BL/6 mice were treated subcutaneously with TGFRt15-TGFRs or 2t 2. 4 days after treatment (TGFRt 15-TGFRs) or 3 days (2 t 2), treated mice were euthanized and single spleen cell suspensions were prepared.The prepared splenocytes were stained with fluorochrome-conjugated anti-CD 4, anti-CD 8 and anti-CD 44 antibodies and CD4 was analyzed by flow cytometry ⁺ T cells or CD8 ⁺ CD44 in T cells ^{High height} Percentage of T cells. The results indicate that TGFRt15-TGFRs and 2T2 up-regulate the expression of memory marker CD44 on cd4+ and cd8+ T cells (fig. 180). These findings indicate that TGFRt15-TGFRs and 2T2 molecules are able to induce differentiation of mouse T cells into memory T cells.

Example 70: improving texture and/or appearance and/or hair

To examine the effect of 2t2 on hair regrowth, the back hair of C57BL6/J mice (jackson laboratory) was first cut short with scissors, then depilatory cream (Nair) was applied to the shaved area for 30 seconds, and then wiped clean. After 4 hours, 2t2 (3 mg/kg, single dose), low dose recombinant IL-2 (25000 IU, consecutive 5 days, 1 dose/day) or PBS was administered subcutaneously. Skin pigmentation of mice associated with hair regeneration was monitored, and photographs were taken and analyzed using Image J software. FIG. 181A shows skin pigmentation of PBS, 2t2 or IL-2 treated mice on day 10 after dehairing. Fig. 181B shows the percent pigmentation of each group of mice at day 10 post-treatment, as analyzed using Image J software. The results indicate that treatment of mice with 2t2 or IL-2 promotes hair regrowth after depilation compared to PBS-treated mice.

The back hair of the C57BL6/J mice (Jackson laboratories) was first shortened with scissors, and then the depilatory cream (Nair) was applied to the shave area for just about 30 seconds, and then wiped clean. After 4 hours, 2t2 (3 mg/kg, single dose), low dose recombinant IL-2 (25000 IU, 5 consecutive days, 1 dose/day) or PBS was administered subcutaneously. Skin pigmentation associated with hair regrowth was monitored in mice and photographed and analyzed using Image J software. FIG. 182 shows skin pigmentation at day 14 after depilation in mice treated with PBS, 2t2 or IL-2. The results indicate that treatment with 2t2 or IL-2 promotes hair regeneration after depilation compared to PBS-treated mice.

Example 71: tissue factor coagulation assay following treatment with single or multiple chain chimeric polypeptides

A set of experiments was performed to assess blood clotting after single or multi-chain chimeric polypeptide treatment. To initiate the coagulation cascade, tissue Factor (TF) binds to factor VIIa (FVIIa) to form TF/FVIIa complex. Then, the TF/FVIIa complex binds Factor X (FX), and FX is converted to FXa.

Factor VIIa (FVIIa) Activity assay

One assay for measuring clotting involves measuring factor VIIa (FVIIa) activity. Such assays require the presence of tissue factor and calcium. TF/FVIIa complex activity can be measured by small substrates or native protein substrates, such as Factor X (FX). Phospholipids are also required for TF/FVIIa activity when FX is used as substrate. In this assay, FVIIa activity was determined using a FVIIa specific chromogenic substrate S-2288 (Diapharma, west Chester, ohio). The color change of the S-2288 substrate can be measured spectrophotometrically and is proportional to the proteolytic activity of FVIIa (e.g., TF/FVIIa complex).

In these experiments, the FVIIa activity of the following groups was compared: 219 amino acid ectodomain of tissue factor domain (TF ₂₁₉ ) A multi-chain chimeric polypeptide having a wild-type tissue factor domain and a multi-chain chimeric polypeptide having a mutated tissue factor domain. Chimeric polypeptides containing mutant tissue factor molecules were constructed with mutations to the TF domain at the following amino acid positions: lys20, ile22, asp58, arg135 and Phe140.

To assess FVIIa activity, FVIIa and TF are combined ₂₁₉ Or contain TF ₂₁₉ Is mixed in all wells of a 96-well ELISA plate in a total volume of 70. Mu.L at equimolar concentration (10 nM). After incubation at 37℃for 10 minutes, 10. Mu.L of 8mM S-2288 substrate was added to start the reaction. The incubation was then maintained at 37℃for 20 minutes. Finally, the color change is monitored by reading the absorbance at 405 nm. OD values for the different TF/VIIa complexes are shown in tables 1 and 2. Table 1 shows TF ₂₁₉ Comparison of 21t15-21s Wild Type (WT) and 21t15-21s mutant (Mut). Table 2 shows TF ₂₁₉ Comparison of 21t15-TGFRs Wild Type (WT) and 21t15-TGFRs mutant (Mut). These data show that TF is contained when chromogenic S-2288 is used as a substrate ₂₁₉ FVIIa activity ratio TF of the multi-chain chimeric polypeptides (e.g., 21t15-21s-WT, 21t15-21s-Mut, 21t15-TGFRS-WT, and 21t 15-TGFRS-Mut) ₂₁₉ Low. Notably, and containing wild type TF ₂₁₉ Comprises TF as compared with the multi-chain chimeric polypeptide of (C) ₂₁₉ The mutated multi-chain chimeric polypeptide shows a much lower FVIIa activity.

TABLE 1 FVIIa Activity

Molecules	OD value at 405nm
		TF 219	0.307
21t15/21S-WT	0.136
		21t15/21S-Mut	0.095

WT: wild type TF ₂₁₉ Mut: TF containing mutations ₂₁ 9。

TABLE 2 FVIIa Activity

Molecules	OD value at 405nm
		CM014	0.345
21t15/TGFRS-WT	0.227
		21t15/TGFRS-Mut	0.100

WT: wild type TF ₂₁₉ Mut: TF containing mutations ₂₁₉ 。

Factor X (FX) activation assay

Another analysis of measuring coagulation involves the activation of measurement Factor X (FX). Briefly, TF/VIIa activates Factor X (FX) to factor Xa (FXa) in the presence of calcium and phospholipids. And TF without transmembrane domain ₂₁₉ In contrast, TF contains the transmembrane domain of TF ₂₄₃ The activity of activating FX to FXa is much higher. TF/VIIa-dependent FX activation was determined by measuring FXa activity using FXa-specific chromogenic substrate S-2765 (Diapharma, west Ohio). The color change of S-2765 can be monitored spectrophotometrically and is proportional to the proteolytic activity of FXa.

In these experiments, FX activation of the multi-chain chimeric polypeptides (18 t15-12s, mouse (m) 21t15, 21t15-TGFRs and 21t15-7 s) was compared to positive controls (inovin) or TF ₂₁₉ A comparison is made. TF is set to ₂₁₉ (or contain TF ₂₁₉ The multiplex chimeric polypeptide)/FVIIa complex is mixed in equimolar concentrations (0.1 nM each) in a round bottom well of a 96-well ELISA plate in a volume of 50. Mu.L, after which 10. Mu.L 180nM FX is added. After incubation at 37 ℃ for 15 min (during which time FX converts to Fxa), 8 μl of 0.5M EDTA (which sequesters calcium, thereby stopping FX activation by TF/VIIa) was added to each well to stop FX activation. Next, 10. Mu.L of 3.2mM S-2765 substrate was added to the reaction mixture. Next, the plate absorbance was measured at 405nm and recorded as absorbance at time 0. The plates were then incubated at 37℃for 10-20 minutes. Color change was monitored by reading 405nm absorbance after incubation. Using chromogenic substratesThe results of FX activation by S-2765 as measured by FXa activity are shown in FIG. 183. In this experiment, recombinant human TF containing lipidation was used ₂₄₃ As a positive control for FX activation. Innovan (inovin) was reconstituted to a TF of about 10nM with purified water ₂₄₃ . Next, a 0.1nM TF/VIIa complex was prepared by mixing an equal volume of 0.2nM FVIIa with 0.2nM minovin. Since nomovin (inovin) exhibits very potent FX activation activity, TF ₂₁₉ And contains TF ₂₁₉ Has very low FX activation activity, thereby confirming TF ₂₁₉ In the TF/FVIIa complex, it is not active for activating the natural substrate FX in vivo.

Prothrombin time test

The third assay to measure coagulation is the Prothrombin Time (PT) test, which measures coagulation activity. Here, PT testing was performed using commercially available normal human plasma (Ci-Trol coagulation control (Ci-Trol Coagulation Control), class I). For standard PT testing, recombinant human TF was tested by addition of lipidated in the presence of calcium ₂₄₃ The coagulation reaction is initiated by inovin (inovin). Clotting times were monitored and reported by a STart PT analyzer (Diagnostics Stago, parsippany, N.J.). By adding 0.2mL of the sample to PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6mM CaCl) ₂ Various dilutions of minovin (inovin) diluted in 0.1% bsa) were injected into a cuvette containing 0.1mL of normal human plasma pre-warmed at 37 ℃ and PT analysis was started. In PT analysis, shorter PT times (clotting times) indicate higher TF-dependent clotting activity, while longer PT (clotting times) indicate lower TF-dependent clotting activity.

As shown in FIG. 184, varying amounts of enovin (e.g., reconstituted with purified water, equivalent to 10nM of lipidated recombinant human TF ₂₄₃ Is considered as 100% of the minovin (innovin)) added to the PT analysis, wherein a lower concentration of TF exhibits a dose response relationship ₂₄₃ Resulting in longer PT times (lower clotting activity). For example, 0.001% of the enovin (inovin) has a PT time greater than 110 seconds, which is almost the same as buffer alone.

In another experiment, the TF ₂₁₉ And a multi-chain chimeric polypeptide for PT testing, said polypeptide comprising: 18t15-12s, 7t15-21s, 21t15-TGFRs-WT and 21t15-TGFRs-Mut. FIG. 185 shows TF ₂₁₉ And contains TF ₂₁₉ Has an extended PT time, indicating very low or no clotting activity.

Studies were also performed to assess whether incubation of the multi-chain chimeric polypeptide (32 dβ or human PBMC) in the presence of other cells carrying the receptor for the cytokine component of the multi-chain chimeric polypeptide would affect clotting time in PT assays. To examine whether cells expressing IL-15 receptor (32D beta cells) or cells expressing IL-15 and IL-21 receptor (PBMC) would bind to IL-15-containing multi-chain chimeric polypeptides to mimic native TF as cellular FVIIa receptor, TF-containing peptides were prepared ₂₁₉ Is diluted in PT assay buffer and is associated with 32D beta cells (at 2X10 ⁵ Individual cells/mL) or PBMCs (at 1x10 ⁵ Individual cells/mL) were pre-incubated together at room temperature for 20-30 minutes. PT analysis was then performed as described above. Figures 186 and 187 show TF mixed with 32D beta cells (figure 186) or PBMCs (figure 187) at 100nM final concentration ₂₁₉ And contains TF ₂₁₉ The PT time of the multi-chain chimeric polypeptide of (a) is prolonged, similar to that of 0.001-0.01% of Innovin (equivalent to 0.1pM to 1.0pM TF) ₂₄₃ ). In the opposite TF ₂₄₃ Percent Activity is expressed as TF-containing ₂₁₉ Is 100,000 to 1,000,000 times less TF-dependent clotting activity than inovin. This demonstrates that TF-containing proteins are present even when the molecules bind to intact cell membrane surfaces, such as 32D beta or PBMC ₂₁₉ Also has very low or no TF-dependent clotting activity.

Example 72: characterization of 7t15-21s137L (long version)

The nucleic acid sequence (including the signal peptide sequence) of the 7t15 construct is as follows (SEQ ID NO: 210):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL 7)

GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT

(human tissue factor 219)

AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

(human IL-15)

AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the 7t15 fusion protein is as follows (SEQ ID NO: 209):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL 7)

DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

(human tissue factor 219)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(human IL-15)

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid sequence (including the signal peptide sequence) of the 21s137L construct is as follows (SEQ ID NO: 331):

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(human IL-21)

CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

(human IL-15Rα sushi Domain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

((G4S) 3 Joint)

GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT

(human CD 137L)

CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including the leader sequence) of the 21s137L fusion protein is as follows (SEQ ID NO: 332):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(human IL-21)

QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

(human IL-15Rα sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

((G4S) 3 Joint)

GGGGSGGGGSGGGGS

(human CD 137L)

REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

The following experiments were performed to assess whether the CD137L moiety in 7t15-21s137L binds to CD137 (4.1 BB) intact. On day 1, 96-well plates were coated overnight with 100. Mu.L (2.5. Mu.g/mL) of R5 (coating buffer) containing GAH IgG Fc (G-102-C, andi organism). On day 2, the plates were washed three times and blocked with 300 μl of 1% bsa in PBS for 2 hours at 37 ℃. 10ng/ml of 4.1BB/Fc (838-4B, andi organism) was added at 100. Mu.l/well for 2 hours at room temperature. After three washes 7t15-21s137L (long version) or 7t15-21s137Ls (short version) were added starting at 10nM, or recombinant human 4.1BBL starting at 180ng/mL at 1/3 dilution followed by incubation overnight at 4 ℃. On day 3, the plates were washed three times and 500ng/mL of biotinylated goat anti-human 4.1BBL (BAF 2295, animate) was applied at 100 μl per well followed by incubation at RT for 2 hours. Plates were washed three times and incubated at 100. Mu.L per well with 0.25. Mu.g/mL HRP-SA (Jackson immunization study) for 30min. Plates were then washed three times and incubated with 100 μl ABTS for 2min at RT. The results were read at 405 nm. As shown in FIG. 188, both 7t15-21s137L (long version) and 7t15-21s137L (short version) can interact with 4.1BB/Fc (dark diamonds and grey squares) as compared to human recombinant 4.1BB ligand (rhCD 137L, light grey star). 7t15-21s137L (long panels) (dark diamonds) interacted better with 4.1BB/Fc than 7t15-21s137L (short panels) (grey squares).

The following experiments were performed to assess whether components IL7, IL21, IL15 and 4.1BBL in 7t15-21s137L (long version) were intact for detection by individual antibodies using ELISA. 96-well plates were coated with 100. Mu.L (4. Mu.g/mL) of R5 (coating buffer) containing anti-TF (human IgG 1) and incubated for 2 hours at RT. Plates were washed 3 times and blocked with 100 μl of PBS containing 1% bsa. Purified 7t15-21s137L (long version) was added starting at 10nM and at 1/3 dilution followed by incubation at RT for 60min. Plates were washed three times and 500ng/mL biotinylated anti-IL 7 (506602, animate) 500ng/mL biotinylated anti-IL 21 (13-7218-81, animate), 50ng/mL biotinylated anti-IL 15 (BAM 247, animate) or 500ng/mL biotinylated goat anti-human 4.1BBL (BAF 2295, animate) were added per well and incubated for 60min at room temperature. Plates were washed three times and incubated at RT for 30min with 0.25. Mu.g/mL HRP-SA (Jackson immunization study) at 100. Mu.L per well. Plates were washed four times and incubated with 100 μl ABTS for 2min at room temperature. Absorbance results were read at 405 nm. As shown in FIGS. 189A-189D, components in 7t15-21s137L (long panel), including IL7, IL21, IL15 and 4.1BBL, were detected by individual antibodies.

The following experiments were performed to evaluate IL15 activity in 7t15-21s137L (long version) and 7t15-21s137L (short version). The ability of 7t15-21s137L (long version) and 7t15-21s137L (short version) to promote proliferation of CTLL2 cells expressing IL2Rαβγ was compared to recombinant IL 15. IL15 dependent CTLL2 cells were washed five times with IMDM-10% FBS and at 2X10 ⁴ Individual cells/wells were seeded into the wells. Serial dilutions of 7t15-21s137L (long version), 7t15-21s137L (short version) or IL15 were added to the cells. Cell in CO ₂ Incubate in incubator at 37℃for 3 days. By at the first20. Mu.L of PrestonBlue (A13261, siemens Feier) was added to each well 3 days, and at CO ₂ Cell proliferation was detected by further incubation at 37℃for 4 hours in the incubator. The raw absorbance at 570-610nm was read in a microtiter plate reader. As shown in FIG. 190, 7t15-21s137L (long version), 7t15-21s137L (short version) and IL15 all promote CTLL2 cell proliferation. EC of 7t15-21s137L (long version), 7t15-21s137L (short version) and IL15 ₅₀ 51.19pM, 55.75pM and 4.947pM, respectively.

Example 73:2t 2-induced Treg cells

Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors (donor 163) were isolated from 5mL whole blood buffy coat by Ficoll Paque Plus (GE 17144003). PBMCs were then lysed with ACK to remove red blood cells. Cells were washed with IMDM-10% fbs and counted. Will be 1.8X10 ⁶ Individual cells (100 μl/tube) were seeded into the flow tube and incubated with 50 μl of decreasing concentrations of 2t2 or IL2 (15000, 1500, 150, 15, 1.5, 0.15 or 0 pM) and 50 μl of pre-stained antibodies (anti-CD 8-BV605 and anti-CD 127-AF 647). Cells were incubated in a water bath at 37℃for 30 min. 200. Mu.L of pre-warmed BD Phosflow fixing buffer I (Cat #557870,Becton Dickinson Biosciences) was added to stop stimulation at 37℃over 10 minutes in a water bath. Cells (4.5X10) ⁵ Individual cells/100 μl) were transferred to V-shaped 96-well plates and centrifuged before permeabilizing with 100 μl of-20 ℃ pre-cooled BD photosflow Perm buffer III (cat#bd Biosciences) for 30 min on ice. The cells are then usedIs washed well 2 times and stained with a set of fluorescent antibodies (anti-CD 25-PE, CD4-PerCP-Cy5.5, CD56-BV421, CD45RA-PE-Cy7 and pSTAT5a-AF 488) to distinguish between different lymphocyte subsets and assess pSTAT5a status. Cells were rapidly centrifuged and resuspended in 200 μl FACS buffer for FACSCelesta analysis. As shown in FIG. 191A, 2t2 of 6pM was sufficient to induce CD4 ⁺ CD25 ^hi T _reg Phosphorylation of Stat5a in cells, whereas induction of phosphorylation of Stat5a in the same lymphocyte population requires 43.11pM of IL-2. In contrast, 2t2Induction of CD4 ⁺ CD25 ^- T _con And CD8 ⁺ T _con Stat5a in the cells was phosphorylated less active than IL2 (fig. 191B) or equal to IL2 (fig. 191C). These results indicate that T in activated human PBMC _reg In terms of 2T2 is superior to IL2, and in terms of human blood lymphocyte pStat5a response, 2T2 exhibits T _reg Selectivity was increased compared to IL-2.

EXAMPLE 74 use of Single chain chimeric Polypeptides to improve Hair growth

The backs Mao Timao of 7 week old C57BL6/J mice were dehaired using commercial dehairing cream. Mice were subcutaneously injected on the same day with either a single dose of 2t2 or a low dose of commercially available recombinant IL-2 and then administered daily for another four days. Untreated mice were used as controls. On day 10, mice were sacrificed and skin sections of shaved areas were prepared. Figures 192A-192E show representative H & E staining of skin sections of C57BL6J mice on day 10 post-dehairing. Fig. 192A shows control mice-depilatory only after shaving, fig. 192B shows mice re-administered low dose IL-2 (1 mg/kg) after depilatory, and fig. 192C-192E show mice re-administered 0.3mg/kg (fig. 192C), 1mg/kg (fig. 192D) and 3mg/kg (fig. 192E) 2t2 after depilatory. Black arrows represent anagen hair follicles which will then extend into the dermis and promote hair growth. Fig. 194 shows the total number of anagen hair follicles counted per 10 fields per treatment group. In summary, the data show that 2t2 molecules allow for an increase in the number of anagen hair follicles compared to dehairing alone. This effect is also dose dependent.

Example 75: differentiation of immune cells into memory-like immune cells

Fresh human leukocytes were obtained from blood banks and CD56 was isolated using Rosetteep/human NK cell reagent (Stem cell technology) ⁺ NK cells. Purity of NK cells>70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 (hundred-in Biotechnology). Cells were counted and at 2x10 ⁶ Individual cells/mL were resuspended in 2mM L-glutamine (sammer life technologies), antibiotics (penicillin, 10,000 units/mL;streptomycin, 10,000 μg/mL; race-mer life technologies) and 10% fbs (sea clone) in RPMI 1640 medium (Ji Bike). Cells (1 mL) were transferred to 24-well flat bottom plates and either untreated or expanded with 7t15-21s+ anti-Tissue Factor (TF) antibody (IgG 1) (50 nM) for 14 days with culture medium. Cells were supplemented with fresh 7t15-21s+ anti-TF antibody (IgG 1) (50 nM) to maintain approximately 1X10 ⁶ Cell density of individual cells/mL.

The non-expanded NK cells of the treated group were used as positive control for the complete DNA methylation level (data not shown). Precipitation of NK cells (1X 10) ⁶ ) And genomic DNA (nDNA) was isolated using QIAamp UCP DNA Micro kit (qiagen). Sodium bisulfite treatment was performed on 500ng of purified nDNA using the EZ DNA methylation guidance kit (Zymo Research) according to the manufacturer's protocol. Bisulfite treatment introduces methylation dependent changes in DNA, in which demethylated cytosines are converted to uracils, while methylated cytosines remain unchanged. Bisulphite treated nDNA (10-50 ng) was used as template for PCR amplification of 228bp regions containing the IFN gamma promoter known to be largely regulated by DNA methylation in T cells (CpG-186 and CpG-54, positions relative to the transcription initiation site TSS) using a Pyromark PCR kit (Kjeger) containing the forward primer IFNG127F (5'-ATGGTATAGGTGGGTATAATGG-3') and the biotinylated reverse primer IFNG355R-bio (Biotin-5'-CAATATACTACACCTCCTCTAACTAC-3') (GENEWIZ). PCR conditions were 48 cycles at 95℃for 15 minutes, 95℃for 30 seconds, 56℃for 30 seconds, 72℃for 60 seconds, and then 72℃for 10 minutes. The integrity and quality of the PCR amplified products were observed on a 1.2% TAE agarose gel. DNA methylation status of these two CpG sites was determined by pyrosequencing, which is the gold standard technique for quantitatively measuring DNA methylation at a single CpG site. At the university of John Hopkins genetic resource core organization (Johns Hopkins University Genetic Resources Core Facility), pyrophosphate sequencing reactions were performed using DNA sequencing primers C186-IFNG135F (5'-GGTGGGTATAATGGG-3') (SEQ ID NO: 333) and C54-IFNG261F (5'-ATTATTTTATTTTAAAAAATTTGTG-3') (SEQ ID NO: 334) specific for CpG sites-186 and CpG sites-54, respectively. Commercially available unmethylated and methyl groups The methylation DNA (Zymo Research) served as a control for DNA methylation. The methylation percentages of the two CpG sites (-186 and-54) per treatment were pooled. The percent difference in DNA methylation was calculated relative to the level of DNA methylation at two CpG sites observed in unexposed NK cells.

Analysis of the DNA methylation status of these two ifnγ CpG sites revealed that the DNA demethylation level was higher in NK cells supported by the 7t15-21s+ anti-TF antibody than in unexposed NK cells (figure 194). These NK cells, which were supported by the 7t15-21s+ anti-TF antibody, showed a difference in DNA methylation (i.e., demethylation) of 47.70% + -11.76 compared to unexposed NK cells. The DNA methylation level of these two IFN gamma CpG sites correlates with increased IFN gamma expression following treatment with 7t15-21s+ anti-TF antibody. These data indicate that long term exposure of NK cells (14 days of culture expansion) combined with a combination regimen of 7t15-21s+ anti-TF antibodies is able to induce DNA demethylation of two hypomethylated ifnγ CpG sites (-186 and-54), and that 7t15-21s+ anti-TF antibodies (IgG 1) are able to reprogram gene expression of ifnγ in an epigenetic manner by DNA demethylation of CpG sites, thus allowing NK cells to interconvert into innate immune memory NK cells.

^CIP1 Example 76: chemotherapy induces p21p21 senescence-associated gene expression in C57BL/6 mice

Chemotherapy-induced p21 in C57BL/6 mice ^CIP1 p21 senescence-associated gene expression. Fig. 203A is a schematic diagram showing a processing scheme. C57BL/6 mice were treated with three doses of chemotherapy Docetaxel (DTX) (10 mg/kg) on day 1, day 4 and day 7. Mice were sacrificed on day 9 and lung and liver tissues were harvested to evaluate senescence markers. FIGS. 203B and 203C show p21 in lung (B) and liver (C) tissues, respectively ^CIP1 Expression of p 21. Lung and liver tissue was homogenized in liquid nitrogen by using a mortar and pestle. The homogenized tissue was transferred to fresh Eppendorf tubes containing 1mL of Trizol (Thermo Fischer). Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Kaiji). Using FAM standard from Thermo ScientificThe pre-designed primer p21 ^CIP1 p21, real-time PCR was performed using CFX96 detection system (Bio-Rad). Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct _Target(s) –Ct _18S 。

As shown in FIGS. 203A-203C, senescence marker p21 was induced in lung and liver tissues of docetaxel-treated mice ^CIP1 p21。

Example 77: immunophenotype and cell proliferation after treatment with IL-15-based agents (day 3 post treatment)

Mouse blood was prepared to evaluate different immune cell subsets after treatment with TGFRt 15-TGFRs. C57BL/6,6 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into the following groups: saline control group (n=6), docetaxel and TGFRt15-TGFRs group (n=6) and docetaxel and IL-15SA group (n=6). IL-15 superagonists (IL-15 SA) were constructed and administered as described previously (Zhu et al, J. Immunol.183 (6): 3598-3607, 2009). Three doses of docetaxel (10 mg/kg) were used to induce aging in mice on days 1, 4 and 7. On day 8, mice were treated subcutaneously with PBS or TGFRt15-TGFRs (3 mg/kg) or IL-15SA (0.2 mg/kg). On day 3 post-treatment, mouse blood was collected from the submandibular vein in EDTA-containing tubes. Whole blood was centrifuged in a microcentrifuge at 3000RPM for 10 minutes to collect plasma. Plasma was stored at-80 ℃, while whole blood was subjected to immunocytophenotype analysis by flow cytometry. Whole blood was lysed in ACK buffer for 5 min at room temperature. Cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To assess different types of immune cells in blood, cells were stained for CD4, CD45, CD8 and NK1.1 (hundred-in biotechnology) at RT for 30 min. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) Washing (at 1500RPM for 5 minutes at room temperature). Cells were treated with permeabilization buffer (Invitrogen) at 4 ℃ for 20 min, then washed with Perm buffer (Invitrogen). Thereafter, cells were stained for intracellular markers (Ki 67) and FoxP3 for 30 min at room temperature. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience). These data indicate that the IL-15 based agents TGFRt15-TGFRs and IL-15SA can stimulate and promote NK and CD8 after docetaxel treatment ⁺ Expansion and proliferation of T cells (figure 204).

Example 78: TGFRt15-TGFRs treatment reduces senescence-associated gene expression in C57BL/6 mice

In the lung and liver of C57BL/6 mice, chemotherapy-induced senescence-associated gene expression was significantly reduced with TGFRt 15-TGFRs. C57BL/6 mice were treated with three doses of chemotherapy docetaxel (10 mg/kg) on day 1, day 4 and day 7. On day 8, docetaxel-treated mice were divided into three groups. The first group received no treatment, the second group received TGFRt15-TGFRs, and the third group received IL-15SA. Saline-treated mice served as controls. TGFRt15-TGFRs was administered at a dose of 3mg/kg, whereas IL-15SA was administered at a dose of 0.2 mg/kg. On day 3 after study drug treatment, mice were sacrificed and lungs and liver were collected. FIGS. 205A-205C show p21 in the lungs (A and B), respectively ^CIP1 Expression of p21 and CD26 and p21 in liver (C) tissue ^CIP1 Expression of p 21. Lung and liver tissue was homogenized in liquid nitrogen by using a mortar and pestle. The homogenized tissue was transferred to fresh Eppendorf tubes containing 1mL of Trizol (zemoeimer). Total RNA was extracted using the RNeasy Mini kit (Kaiji # 74106) according to the manufacturer's instructions. 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Kaiji). Pre-designed primer p21 using FAM labeling from Thermo Scientific ^CIP1 p21 and CD26, real-time PCR was performed using CFX96 detection system (Bio-Rad). Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Every time relative to 18S rRNAExpression of the individual target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct _Target(s) –Ct _18S 。

As shown in fig. 205A-205C, a marker of therapy-induced senescence p21 in lung and liver tissues of mice treated with TGFRt15-TGFRs ^CIP1 p21 was significantly reduced. The therapy-induced senescence marker CD26 was also significantly reduced in lung tissue of mice treated with TGFRt 15-TGFRs.

Example 79: immunophenotype after IL-15 based agent treatment

Mouse blood was prepared to evaluate different immune cell subsets after treatment with IL-15 based agents: TGFRt15-TGFRs, IL-15 superagonists (IL-15 SA) and fusion of IL-15 with D8N mutants that knocked out IL-15 activity (TGFRt 15 x-TGFRs). C57BL/6,6 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into groups (n=6/group) and treated with the following method: 1) PBS (saline) control, 2) docetaxel, 3) docetaxel and TGFRt15-TGFRs, 4) docetaxel and IL-15SA, 5) docetaxel and IL-15 mutant (TGFRt 15 x-TGFRs) and 6) docetaxel and IL-15 super-agonist (IL-15 SA) plus TGFRt15 x-TGFRs. Three doses of docetaxel (10 mg/kg) were used to induce aging in mice on days 1, 4 and 7. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, IL-15SA or a combination of the above. TGFRt15-TGFRs and TGFRt15-TGFRs are administered at a dose of 3mg/kg, whereas IL-15SA is administered at a dose of 0.05 mg/kg. On day 3 after study drug treatment, mouse blood was collected from the submandibular vein into EDTA tubes. Whole blood was centrifuged in a microcentrifuge at 3000RPM for 10 minutes to collect plasma. Plasma was stored at-80 ℃, while whole blood was subjected to immunocytophenotype analysis by flow cytometry. Whole blood was lysed in ACK buffer for 5 min at 37 ℃. Cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To assess different types of immune cells in blood, cells were stained for CD4, CD45, CD19, CD8 and NK1.1 (hundred-in biotechnology) at Room Temperature (RT) for 30 minutes. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. Cells were treated with permeabilization buffer (England Life technologies) at 4℃for 20 min, then washed with Perm buffer (England Life technologies). Thereafter, the cells were stained for intracellular markers (Ki 67) for 30 minutes at RT. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience) (fig. 206 and 207).

These data indicate that the IL-15 based agents TGFRt15-TGFRs and IL-15SA can stimulate and promote NK and CD8 after docetaxel treatment ⁺ Expansion and proliferation of T cells. Fusion proteins lacking IL-15 activity (i.e., TGFRt15 x-TGFRs) were devoid of NK and CD8 observed ⁺ An increase in T cell expansion and proliferation.

^CIP1 Example 80: assessment of senescence markers p21 p21 and CD26 in lung and liver tissue

Markers of cellular senescence were assessed in tissues of normal mice after chemotherapy and administration of study treatments. C57BL/6,6 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into six groups and treated as follows: 1) PBS (saline) control (n=5), 2) docetaxel (n=8), 3) docetaxel and TGFRt15-TGFRs (n=8), 4) docetaxel and IL-15SA (n=8), 5) docetaxel and IL-15 mutant (TGFRt 15-TGFRs) (n=8) and 6) docetaxel and IL-15 super-agonist (IL-15 SA) plus TGFRt15-TGFRs (n=6). Three doses of docetaxel (10 mg/kg) were used to induce aging in mice on days 1, 4 and 7. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, IL-15SA or a combination of the following. TGFRt15-TGFRs and TGFRt15-TGFRs are administered at a dose of 3mg/kg, whereas IL-15SA is administered at a dose of 0.05 mg/kg. Mouse tissues were prepared to evaluate different senescence markers. Mice were euthanized on day 7 after study drug treatment, liver and lung tissue was collected and stored in liquid nitrogen in 1.7mL Eppendorf tubes. Samples were homogenized in liquid nitrogen by using a mortar and pestle. The homogenized tissue was transferred to a container containing 1mL of Trizol (Sieimer's fly) World) in fresh Eppendorf tubes. Total RNA was extracted using the RNeasy Mini kit (QIAGER# 74106) according to the manufacturer's instructions and 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (QIAGER). Real-time PCR was performed with a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers from Thermo Scientific. Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct _Target(s) –Ct _18S 。

As shown in FIGS. 208A-208C, senescence markers p21 and CD26 were found in the lungs of docetaxel-treated mice [ respectively (A) and (B) ]]Is induced in p21 ^CIP1 p21 is induced in liver (C) tissue. Mice treated with a combination of TGFRt15-TGFRs, IL-15SA and TGFRt15-TGFRs mutants, senescence marker p21 in the lung ^CIP1 p21 and CD26 and senescence marker p21 in liver ^CIP1 p21 decreases. However, TGFRt15 x-TGFRs mutant treated mouse lungs failed to eliminate senescence markers in these tissues. These results indicate that IL-15 activity is important for the clearance of TIS senescent cells.

EXAMPLE 81 immunophenotype after TGFRt15-TGFRs treatment

Mouse blood was prepared to evaluate different immune cell subsets after treatment with TGFRt 15-TGFRs. C57BL/6, 76 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). On day 0, mice were treated subcutaneously with PBS or with TGFRt15-TGFRs at a dose of 3 mg/kg. On day 4 after the first dose of study treatment, mouse blood was collected from the submandibular vein in EDTA-containing tubes. Whole blood was centrifuged in a microcentrifuge at 3000RPM for 10 minutes to collect plasma. Plasma was stored at-80 ℃, while blood was subjected to immunocytophenotype analysis by flow cytometry. Whole blood was lysed in ACK buffer for 5 min at room temperature. Cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To assess different types of immune cells in blood, cells were stained for CD4, CD45, CD19, CD8 and NK1.1 (hundred-in biotechnology) at Room Temperature (RT) for 30 minutes. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at RT. Cells were treated with permeabilization buffer (England Life technologies) at 4℃for 20 min, then washed with Perm buffer (England Life technologies). Thereafter, the cells were stained for intracellular markers (Ki 67) for 30 minutes at RT. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in figure 195, CD8 4 days after administration of the first dose of TGFRt15-TGFRs ⁺ Percentage of T cells and CD8 as measured by Ki67 ⁺ T cell proliferation increases significantly. We also observed an increase in NK cells and proliferation of NK cells, as shown in figure 196. We observed CD19 after administration of the first dose of TGFRt15-TGFRs ⁺ The cells were significantly reduced. These results indicate that subcutaneous administration of a single dose of TGFRt15-TGFRs can stimulate immune cells (e.g., CD8 ⁺ T cells and NK cells) proliferate in the blood of aged mice.

Example 82: TGFRt15-TGFRs reduces aging related beta-Gal from liver and lung tissue

Mouse liver and lung were prepared to evaluate β -gal associated with aging in tissues after treatment with TGFRt 15-TGFRs. C57BL/6, 76 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). On day 0 and 10, mice were treated subcutaneously with PBS or with TGFRt15-TGFRs at a dose of 3 mg/kg. On day 7 after the second dose of study treatment, mice were euthanized, liver and lungs were harvested, homogenized in PBS containing 2% PBS, and filtered in a 70 micron filter to obtain a single cell suspension. The cells were centrifuged and then resuspended in a 14mL round bottom tube containing 0.5mg/mL collagenase IV and 0.02mg/mL DNAs e 5mL RPMI. The cells were then shaken on a rotary shaker for 1 hour at 37 ℃. Cells were washed twice with RPMI. Cells were counted and at 2x10 ⁶ 2mL of complete medium (RPMI 1640 (Ji Bike), supplemented with 2mM L-glutamine (Semer Life technologies), penicillin (Semer Life technologies), streptomycin (Semer Life technologies) and 10% FBS (sea clone)) resuspended in 24 well flat bottom plates and incubated at 37℃in 5% CO ₂ Is cultured for 48 hours. Cells were harvested and washed once in warm complete medium at 1000rpm for 10 minutes at room temperature. The cell pellet was resuspended in 500. Mu.L of fresh medium, containing 1.5. Mu.L of senescence dye per tube. The cells were then incubated at 37℃with 5% O ₂ For 1-2 hours and washed 2 times (2X) with 500. Mu.L of wash buffer. The cell pellet was resuspended in 500. Mu.L of wash buffer and analyzed immediately by flow cytometry (Celesta-BD bioscience).

As shown in FIG. 197, aging-related beta-gal 7 days after administration of the second dose of TGFRt15-TGFRs ⁺ The percentage of cells decreases. These results indicate that TGFRt15-TGFRs can reduce β -gal associated with aging in aged mouse tissues.

^CIP1 Example 83: markers of aging CD26, IL-1 alpha, p16.sup.INK4 and p21 in kidney, skin, liver and lung tissue

The kidneys, skin, liver and lungs of mice were harvested to assess the senescence markers CD26, IL-1 alpha, p16 and p21 by quantitative PCR in tissues treated with TGFRt15-TGFRs or PBS control. C57BL/6, 76 week old mice were purchased from Jackson laboratories. Mice were placed in a temperature and light controlled environment for one week prior to any study. Mice were divided into two groups: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). On day 0 and 10, mice were treated subcutaneously with PBS or with TGFRt15-TGFRs at a dose of 3 mg/kg. On day 7 after the second dose of study treatment, mice were euthanized, kidneys, skin, liver and lungs were harvested and stored in liquid nitrogen in 1.7mL Eppendorf tubes. Samples were homogenized in liquid nitrogen by using a mortar and pestle. The homogenized tissue was transferred to a container containing 1mL of Trizol (Sieimer's feichil)) Is prepared in a fresh Eppendorf tube. Total RNA was extracted using the RNeasy Mini kit (QIAGER# 74106) according to the manufacturer's instructions and 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (QIAGER). Real-time PCR was performed with a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers from Thermo Scientific. Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct _Target(s) –Ct _18S 。

As shown in FIGS. 198-201, there was no difference between senescence markers CD26 and IL-1α, but p21 ^CIP1 Reduced expression in liver (panel 198), lung (panel 201) and skin (panel 200) is shown in TGFRt15-TGFRs treated mice. In the kidney (FIG. 199), p21 7 days after administration of the second dose of TGFRt15-TGFRs ^CIP1 And the il1α markers were significantly reduced in both aged mice.

Example 84: beta-Gal staining of kidney tissue by histology

The mouse kidneys were prepared to evaluate the senescence marker β -gal in kidney tissue after treatment with TGFRt 15-TGFRs. C57BL/6, 76 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). On day 0 and 10, mice were treated subcutaneously with PBS or with TGFRt15-TGFRs at a dose of 3 mg/kg. On day 7 after the second dose of study treatment, mice were euthanized and kidneys were harvested, and half of the kidney tissue was embedded in a frozen section embedding mold (tissue-tek cyromaldes) containing OCT compound. The frozen section embedding mold containing the tissue was immediately frozen in the vapor phase of liquid nitrogen. The samples were further processed to cut into 4-8um thick cryostat sections (Lecia Cm 1800 cryostat) and mounted on superfrost plus slides. Slides with sections were processed according to manufacturer's protocol for senescence beta-galactosidase staining kit (Cell Signaling). Tissue sections were observed under a microscope.

As shown in graph 202, senescence-associated β -gal was observed in TGFRt15-TGFRs treated mice compared to control mice (n=3) ⁺ The number of cells is reduced. These results indicate that TGFRt15-TGFRs treatment can reduce β -gal associated with aging in aged mouse tissues.

Example 85: production of TGFRt15-TGFRs fusion proteins

A fusion protein complex was generated comprising TGFR/IL15RαSu and TGFR/TF/IL-15D8N fusion proteins (FIGS. 209 and 210). Human TGF-beta receptor (TGFR), IL-15 alpha receptor sushi domain (IL 15 RaSu), tissue Factor (TF) and IL-15 (IL 15D 8N) sequences with D8N mutants were obtained from GenBank website and DNA fragments of these sequences were synthesized by Jin Weizhi. Specifically, a structure was constructed in which the TGFR sequence was linked to the N-terminal coding region of IL15RaSu, the TGFR sequence was linked to the N-terminal of tissue factor 219, and then to the N-terminal coding region of IL-15D 8N.

The nucleic acid sequence (including the signal peptide sequence) of the TGFR/IL15RaSu construct is as follows:

(Signal peptide)

ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

(Single chain human TGF-beta receptor II homodimer)

ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(Sushi domain of IL15 receptor alpha chain)

ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

The nucleic acid sequence (including the signal peptide sequence) of the TGFR/TF/IL15D8N construct is as follows:

(Signal peptide)

ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC

(Single chain human TGF-beta receptor II homodimer)

ATCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

(human tissue factor 219)

TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA

(human IL-15D 8N)

AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT

The amino acid sequence (including the signal peptide sequence) of the TGFR/IL15RaSu fusion protein is as follows (SEQ ID NO:):

(Signal peptide)

MKWVTFISLLFLFSSAYS

(Single chain human TGF-beta receptor II homodimer)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(human IL-15 receptor. Alpha. Sushi Domain)

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence (including the signal peptide sequence) of the TGFR/TF/IL15D8N fusion protein is as follows (SEQ ID NO:):

(Signal peptide)

MGVKVLFALICIAVAEA

(Single chain human TGF-beta receptor II homodimer)

IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

(tissue factor)

SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

(IL-15D8N)

NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The TGFR/IL15RαSu and TGFR/TF/IL-15D8N constructs were cloned into modified retroviral expression vectors as described previously (Hughes MS, yu YY, dudley ME, zheng Z, robbins PF, li Y et al). The expression vector was transfected into CHO-K1 cells. Co-expression of both constructs in CHO-K1 cells allows the formation and secretion of soluble TGFR/IL15RαSu-TGFR/TF/IL-15D8N protein complexes (called TGFRt15 x-TGFRs) which can be purified by anti-TF antibody affinity.

Example 86: TGFRt15-TGFRs and TGFRt15-TGFRs binding Activity to TGF-beta 1 and LAP

The binding activity of TGFRt15-TGFRs to TGF-beta 1 and LAP was determined by ELISA. TGFRt15-TGFRs (5 mg/mL) was used to capture titrated TGF- β1 (labeled TGF- β1, hundred-in biotechnology) and latency-related peptides of TGF- β1 (LAP, andi organism). TGF-. Beta.1 was detected by biotinylated anti-TGF-. Beta.1 (0.2 mg/mL, andi organism), by biotinylated anti-LAP (0.2 mg/mL, andi organism), followed by peroxidase-coupled streptavidin (Jackson immunoresearch laboratory). 2,2' -Di-N-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, surmotics IVD) was used as substrate and measured by a plate reader. As shown in fig. 211A, the results demonstrate that TGFRt15-TGFRs bind similarly to TGF- β1 and LAP and are stronger than Fc fusions.

The binding activity of TGF-beta 1 receptor/Fc fusion to TGF-beta 1 and LAP was determined by ELISA. Commercial TGF-beta 1 receptor II-Fc fusions (TGFRII/Fc) were used to compare the binding activity of TGFRt15-TGFRs to TGF-beta 1 and LAP. TGFRII/Fc (5 mg/mL, andi organism) was used to capture titrated TGF-. Beta.1 and LAP. The other procedure is the same as described above. As shown in FIG. 211B, the results demonstrate that TGFRII/Fc binds similar to TGF-. Beta.1 and LAP, with binding comparable to TGFRt15-TGFRs and stronger than Fc fusions.

TGFRt15-TGFRs and TGFRt15-TGFRs binding Activity to TGF-beta 1 and LAP

TGFRt15-TGFRs and TGFRt15-TGFRs (10 mg/mL) were used to capture titrated TGF-beta 1LAP. The other procedure is the same as described above. As shown in figures 211C and D, the results demonstrate that TGFRt15 x-TGFRs bind to TGF- β1 and LAP similarly, with binding comparable to TGFRt15-TGFRs and stronger than Fc fusions.

TGFRt15-TGFRs and TGFRt15-TGFRs binding to CTLL-2 cells

IL-2 dependent CTLL-2 cells were stained with TGFRt15-TGFRs (50 nM), 7t15-21s (50 nM, IL-7-TF-IL15 and IL-21-IL-15 RaSu) (as control fusion molecules, without TGF-. Beta.1 receptor II), PBS (as negative control) for 60 min and probed by biotinylated secondary antibodies (anti-TF: anti-human tissue factor, HCW Biologics (HCW Biologics) and anti-TGFR: anti-TGF-. Beta.receptor II: andi organism) and then by R-phycoerythrin-streptavidin (Jackson immunoresearch laboratory). The Mean Fluorescence Intensity (MFI) of the staining was measured by flow cytometry. As shown in FIG. 211E, the results demonstrate that TGFRt15-TGFRs bind to CTLL-2 cells significantly better than other molecules because of the IL-15 mutant, TGFRt15 x-TGFRs is lower than TGFRt15-TGFRs. However, binding of 7t15-21s to CTLL-2 cells can be detected with anti-TF instead of anti-TGFR.

Example 87: cell-based assays to determine TGFRt15-TGFRs and the biological activity of TGFRt15-TGFRs

TGFRt15-TGFRs and TGFRt 15-TGF- β1 blocking activity of TGFRs.

HEK-Blue TGF-beta cells (InvivoGen) were incubated in IMDM-10 containing titrated TGFRt15-TGFRs, TGFRt15 x-TGFRs, and TGFRII/Fc as controls in the presence of TGF-beta 1 (0.1 nM, hundred-Biotech). TGFRII/Fc is a commercial TGF-beta 1 receptor II-Fc fusion (Andi organism). After 24 hours of incubation, the culture supernatant was mixed with QUANTI-Blue (InvivoGen) and incubated for 1-3 hours. OD620 values were measured by a microplate reader. As shown in figure 212A, TGFRt15-TGFRs and TGFRt15-TGFRs have the same TGF- β1 blocking activity. In contrast, TGF- β1 blocking activity of TGFRII/Fc (ic50=470.2 pM) was about 10-fold lower than TGFRt15-TGFRs (ic50=43.2 pM) or TGFRt15-TGFRs (45.2 pM). Blocking activity was calculated using GraphPad Prism 7.04.

IL-15 Activity of TGFRt15-TGFRs and TGFRt15-TGFRs

IL-15 dependent 32D beta cells were cultured in IMDM-10 containing titrated TGFRt15-TGFRs, and IL15 as a control. After 2 days WST-1 (Feishan technology) was added and OD450 values were measured by means of a microplate reader. As shown in fig. 212B, IL-15 bioactivity of TGFRt15-TGFRs (ec50=1641 pM) was 20-fold lower than IL-15 itself (ic50=81.8 pM). As expected, TGFRt15-TGFRs had no detectable IL-15 activity. IL-15 activity was calculated using GraphPad Prism 7.04.

Reverse of TGFRt15-TGFRs inhibition of TGF-beta growth of CTLL-2

TGF-beta includes three isoforms (TGF-beta 1, TGF-beta 2, and TGF-beta 3), with similar biological functions. The present study uses CTLL-2 cells to compare the biological blocking activity of TGFRt 15-TGFRs. TGFRt 15. Times. -TGFRs are very similar in structure to TGFRt15-TGFRs, but cannot be used in this example due to the IL-15 activity of TGFRt 15-TGFRs. CTLL-2 cells were incubated with titrated mouse IL-4 (bai biotechnology), TGF- β (5 ng/ml, TGF- β1 (bai biotechnology), TGF- β2, β3 (anitbox)) and TGFRt15 x-TGFRs (21 nm; TGFRt15 x-TGFRs: TGF- βmolar ratio = 100:1) in RPMI-10 for 5 days. After adding PrestoBlue (femal technology) to the culture of the last day, cell proliferation was measured by an enzyme-labeled instrument (OD _570-600 Values). Fig. 212C shows that all three TGF- β similarly inhibited IL-4 induced CTLL-2 growth in the absence of TGFRt15 x-TGFRs. Fig. 212D shows that TGFRt15 x-TGFRs (21 nm; TGF- β: TGFRt15 x-TGFRs molar ratio = 1:100) significantly reverse TGF- β1 and TGF- β3 inhibition of IL-4 induced CTLL-2 cell growth, in contrast to TGFRt15 x-TGFRs with minimal reverse TGF- β2 inhibitory activity.

Example 88: stability of TGFRt15-TGFRs

The stability of TGFRt15-TGFRs was examined by ELISA. TGFRt15-TGFRs were pre-incubated in RPMI medium containing 50% human serum for 10 days at 4 ℃, room Temperature (RT) or 37 ℃. The IL-15 domain and TGF-beta RII domain of TGFRt15-TGFRs were evaluated by ELISA. anti-TF antibodies (HCW Biologics) are used to capture TGFRt15-TGFRs molecules, while biotinylated anti-IL-15 (animate) is used to detect IL-15 domains and biotinylated anti-tgfbetarii (animate) is used to detect tgfbetarii domains. Biotinylated detection antibodies were probed by peroxidase-streptavidin (jackson immunoresearch laboratory). 2,2' -diaza-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, surmotics IVD) was used as substrate and OD405 was measured by an enzyme-labeled instrument. As shown in figures 213A and B, the results indicate that there was no significant change in the domain of TGFRt15-TGFRs after incubation at 4 ℃, RT or 37 ℃ for 10 days. These findings indicate that the IL-15 domain and TGFβRII domain of TGFRt15-TGFRs remain intact when incubated with human serum under evaluation conditions.

Determination of stability of TGFRt15-TGFRs biological Activity Using cell-based assays

TGFRt15-TGFRs were pre-incubated with 50% human serum in RPMI-10 for 10 days at 4 ℃, room Temperature (RT) or 37 ℃. TGFRt15-TGFRs TGF- β1 neutralization activity was obtained using HEK-Blue TGF- β1 cells (TGF- β1 activity reporter cell line, invivoGen). HEK-Blue TGF-beta cells were incubated in IMDM-10 containing titrated TGFRt15-TGFRs in the presence of TGF-beta 1 (0.1 nM). After 24 hours of incubation, the culture supernatant was mixed with QUANTI-Blue (InvivoGen) and incubated for 1-3 hours. OD620 values were measured by a microplate reader. As shown in FIG. 213C, the results indicate that TGFRt15-TGFRs has no change in TGF-. Beta.1 neutralization activity after incubation in human serum at 4 ℃, RT or 37 ℃ for 10 days. IL-15 activity of TGFRt15-TGFRs was assessed with IL-15 dependent 32D beta cells. 32D beta cells were cultured in IMDM-10 containing titrated TGFRt 15-TGFRs. After 2 days WST-1 (England Life technologies) was added and OD450 values were measured by a microplate reader. As shown in FIG. 213D, the results indicate that there is no change in IL-15 activity of TGFRt15-TGFRs after incubation in human serum for 10 days at 4 ℃, RT or 37 ℃.

Example 89: TGFRt15-TGFRs and TGFRt15-TGFRs TGF-beta 1 immunosuppression of human NK cells and PBMC Reversion of production

According to the StemCell specification, rosetteep is used ^TM Human NK cell enriched mixture (StemCell) human NK cells were purified from blood buffy coat (4 donors, one blood) and PMBC was isolated from blood buffy coat (6 donors) using Ficoll-Paque (sigma-aldrich) density centrifugation. NK cells and PBMC were cultured in RPMI-10 containing IL-15 (10 ng/mL, peproTech) and/or TGF-. Beta.1 (10 ng/mL, hundred-Biotech), TGFRt15-TGFRs (42 nM or 4.2 nM) or TGFRt15-TGFRs (42 nM or 4.2 nM) for 3 days. Cultures were collected and used for the following assays: cell-mediated cytotoxicity assays (fig. 214A and B) and flow cytometry for intracellular granzyme B (fig. 214C and D) and interferon gamma (ifnγ, fig. 214E and F) (flow cytometry analyses).

In cell-mediated cytotoxicity assays, cultured NK cells and PBMCs were used as effector cells, while K562 tumor cells (ATCC) were used as target cells. The mixture of effector cells and K562 tumor cells was incubated in RPMI-10 for 4 hours at 37 ℃, E: T ratio = 4:1 for NK cells (fig. 214A) or E: T ratio = 20:1 for PBMCs (fig. 214B). The level of dead K562 cells was determined by flow cytometry. As shown in fig. 214A and B, the results demonstrate that significantly fewer K562 target cells die in the presence of TGF- β1 compared to the observed media control culture, indicating that TGF- β1 inhibits cytotoxicity of immune cells. However, in the presence of TGF- β1 and TGFRt15-TGFRs or TGFRt15 x-TGFRs, there were significantly more K562 target cells that died compared to cultures incubated with TGF- β1 alone observed. These findings indicate that TGFRt15-TGFRs and TGFRt15 x-TGFRs significantly reduce TGF- β1 immunosuppression and enhance cytotoxicity of human NK cells and PBMCs on K562 target cells in a concentration-dependent manner. In addition, IL-15 activity of TGFRt15-TGFRs further enhanced cytotoxicity of human NK cells and PBMC compared to the activity of TGFRt 15-TGFRs.

Cultured NK cells and PBMC was stained with fluorochrome-labeled anti-CD 56 and anti-CD 16 human NK cell surface markers, followed by fluorochrome-labeled granzyme B and IFN gamma intracellular molecules (hundred-in biotechnology). Purified NK cells and gated NK cells (CD 56) in PBMC cultures by flow cytometry analysis ⁺ And/or CD16 ⁺ ) Granzyme B and interferon gamma expression (MFI: average fluorescence intensity). As shown in figures 214C and D, granzyme B (figures 214C and 214D) and ifnγ (figures 214E and 214F) expression in cultured NK cells was significantly less in the presence of TGF- β1 than that observed in cells cultured in medium alone, indicating that TGF- β1 inhibited immune cell activation. However, granzyme B and ifnγ expressing NK cell cultures were significantly more in the presence of TGF- β1 and TGFRt15-TGFRs or TGFRt15 x-TGFRs than observed in cells cultured in TGF- β1 alone. The minimum concentration of TGFRt15-TGFRs effect on granzyme B and ifnγ expression was 4.2nM. These findings indicate that TGFRt15-TGFRs and TGFRt15-TGFRs significantly enhance granzyme B and ifnγ expression of human NK cells in a concentration-dependent manner by the activity of IL-15 and tgfbrii domains.

Example 90: half-life of TGFRt15-TGFRs in C57BL/6 mice

The pharmacokinetics (half-life, t 1/2) of TGFRt15-TGFRs were evaluated in female C57BL/6 mice. Mice were treated subcutaneously with a dose of 3mg/kg of TGFRt15-TGFRs. Mouse blood was collected from the tail vein at various time points, and serum was prepared. The concentration of TGFRt15-TGFRs in mouse serum was determined by ELISA. anti-TF antibodies (anti-human tissue factor antibodies generated in HCW Biologics) are used to capture TGFRt15-TGFRs molecules, while biotinylated anti-tgfbrii (animalcule) is used to detect tgfbrii domains. Biotinylated detection antibodies were probed by peroxidase-streptavidin (jackson immunoresearch laboratory). 2,2' -diaza-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, surmotics IVD) was used as substrate and OD405 was measured by an enzyme-labeled instrument. As shown in FIG. 215, the half-life of TGFRt15-TGFRs in C57BL/6 mice calculated using GraphPad Prism 7.04 was 18.22 hours.

Example 91: C5C 5Toxicity of TGFRt15-TGFRs in 7BL/6 mice

A single dose of TGFRt15-TGFRs (50-400 mg/kg) was subcutaneously injected into C57BL/6 female mice (7 weeks old, n=4). Mice body weight was measured as shown in figure 216 and clinical signs (mortality, morbidity, fur fold, humpback posture, somnolence, etc.) were assessed during the experiment. Mice receiving 200mg/kg or 400mg/kg of TGFRt15-TGFRs showed lower activity 6-8 days after treatment, with no other obvious clinical symptoms. Compared to the PBS group, 200mg/kg or 400mg/kg of TGFRt15-TGFRs resulted in weight loss in mice, especially at day 7 after treatment (p < 0.05). The affected mice gradually recovered after 10 days without death or morbidity. As shown in FIG. 216, these findings indicate that C57BL/6 mice can tolerate a single dose of TGFRt15-TGFRs up to 100 mg/kg.

Example 92: antitumor Activity of TGFRt15-TGFRs in C57BL/6 murine melanoma model

Mouse B16F10 melanoma cells were subcutaneously injected into C57BL/6 mice (jackson laboratory) to establish a mouse melanoma model. Four days after tumor cell injection, mice were divided into different groups to receive the following immunotherapy: group 1: PBS vehicle control; group 2: anti-tumor antibody TA99 alone (10 mg/kg) control; group 3: TA99 in combination with IL-15SA (0.05 mg/kg); group 4: TA99 in combination with TGFRt15-TGFRs (4.93 mg/kg,0.05mg/kg IL-15SA equivalent IL-15 activity); group 5: TA99 in combination with TGFRt15-TGFRs (4.93 mg/kg, IL-15D8N mutant without IL-15 activity). Tumor volumes were measured and calculated using the following formula: the length x width/2 formula. As shown in figure 217, the results demonstrate that mice receiving the anti-tumor antibody TA99 in combination with TGFRt15-TGFRs or IL15SA had significantly smaller tumors on day 11 post tumor inoculation compared to PBS, TA99 antibody alone and TA99 and TGFRt15-TGFRs group (p < 0.05). There were no significant differences between groups 1, 2 and 5 and between groups 3 and 4. These findings indicate that IL-15 activity of TGFRt15-TGFRs is important for the anti-tumor activity of TGFRt 15-TGFRs.

Example 93: model of pulmonary fibrosis-treatment with TGFRt15-TGFRs

Inflammation and fibrotic lung disease (including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and cystic fibrosis) are leading causes of death, with limited treatment options. In addition, various therapies can lead to lung injury side effects, leading to pulmonary fibrosis. For example, pulmonary toxicity occurs in about 10% of cancer patients receiving bleomycin chemotherapy. These side effects have led to the use of bleomycin therapy in rodents to mimic pulmonary fibrosis for studying mechanisms involved in fibrosis formation and evaluating potential therapies. To evaluate the activity of TGFRt15-TGFRs in this model, nine week old C57Bl6/j male mice were administered 50 μl bleomycin (2.5 mg/kg, single dose) via the oropharyngeal route. On day 17 after bleomycin treatment, mice were subcutaneously injected with TGFRt15-TGFRs (3 mg/kg). Mice were sacrificed on day 28 after bleomycin treatment. The lungs were isolated and left lung homogenized and the hydroxyproline content of 100 μl of the homogenate was determined as a measure of collagen deposition using a commercially available kit according to the manufacturer's instructions. Data are expressed as how much μg hydroxyproline per gram of lung. As shown in figure 218, the results demonstrate that TGFRt15-TGFRs therapy significantly reduced collagen deposition (i.e., fibrosis) in the lungs of bleomycin-treated mice.

Example 94: in vivo characterization of TGFRt15-TGFRs and TGFRt15-TGFRs activity

Leptin-deficient ob/ob mice have been shown to be protected from obesity and diabetes by blocking TGF- β/Smad3 signaling. To assess whether TGFRt15-TGFRs or TGFRt15-TGFRs can protect mice from obesity and diabetes by blocking TGF- β/Smad3 signaling, leptin receptor-deficient db/db mouse strains (bks.cg Dock7m ^+/+ Leprdb/J) was used for the study. Six weeks old db/db mice were divided into three groups (n=8 per group). Mice were subcutaneously injected with TGFRt15-TGFRs, TGFRt15-TGFRs or PBS at a dose of 3mg/kg. Blood was collected via the submaxillary vein on day 4 post injection after 20 hours of fasted mice. Immediately after blood withdrawal, fasting blood glucose was measured with a OneTouch UltraMini glucometer. As shown in FIG. 219, both TGFRt15-TGFRs and TGFRt15-TGFRs significantly reduced fasting blood glucose levels.

Plasma TGF-beta 1-3 levels were assessed to determine the cause of treatment-related fasting blood glucose reduction in db/db mice. Four days after treatment, plasma was isolated and 30. Mu.L of plasma was sent to EVE Technologies (Canada, alberta, calgari) and TGF-beta 1-3 levels were assessed by TGF-beta 3-Plex (TGFB 1-3) assay. As shown in figures 220A-C, both TGFRt15-TGFRs and TGFRt15-TGFRs completely deplete plasma tgfβ1 (figure 220A), partially reduce tgfβ2 (figure 220B) and have no effect on tgfβ3 (figure 220C).

The lymphocyte subpopulation was evaluated to determine the cause of treatment-related fasting blood glucose reduction in db/db mice. Four days after treatment, whole blood cells (50 μl) were treated with ACK (ammonium-potassium chloride) lysis buffer to lyse erythrocytes. Lymphocytes were then stained with PE-Cy 7-anti-CD 3, BV 605-anti-CD 45, perCP-Cy5.5-anti-CD 8a, BV 510-anti-CD 4 and APC-anti-NKp 46 (all antibodies from the hundred-in biotechnology) to assess T-cell and NK-cell populations. Cells were further permeabilized and fixed with the electronic biotechnology (eBioscience) Foxp 3/transcription factor staining buffer set (Cat#00-5523-00, siemens) and stained with AF 700-anti-Ki 67 and FITC-anti-granzyme B in the electronic bioscience (eBioscience) permeabilization buffer (Cat#00-8333-56, siemens) to assess proliferation and activation of T cells and NK cells. Another group of lymphocytes was stained with PE-Cy 7-anti-CD 3, BV 605-anti-CD 45, BV 510-anti-CD 4 and apc-Cy 7-anti-CD 25, then permeabilized and fixed with the electronic biotechnology (eBioscience) Foxp 3/transcription factor staining buffer group (Cat#00-5523-00, siemedas) and stained with PE-anti-Foxp3 in the electronic biotechnology (eBioscience) permeabilization buffer (Cat#00-8333-56, siemedas) to assess Treg cell populations.

TGFRt15-TGFRs increased NK cell populations (FIG. 221A) and CD8 ⁺ T cell population (panel 221D), stimulated NK cells (panel 221B) and CD8 ⁺ Proliferation of T cells (fig. 221E) and activation of NK cells (fig. 221C). TGFRt15-TGFRs had no effect on either cell population (fig. 221A-E). TGFRt15-TGFRs and TGFRt15-TGFRs vs. CD4 ⁺ T cells, CD19 ⁺ B cells and CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Treg cells had no effect.

In short, inIn db/db mice, both TGFRt15-TGFRs and TGFRt15-TGFRs reduced fasting blood glucose levels, and both TGFRt15-TGFRs and TGFRt15-TGFRs completely depleted plasma tgfβ1. However, only TGFRt15-TGFRs activated NK cells and enhanced CD8 ⁺ T cell and NK cell proliferation. Based on these results, depletion of tgfβ1 may be associated with a decrease in fasting blood glucose, suggesting that blocking TGF- β/Smad3 signaling plays a role in obesity and diabetes prevention in ob/ob mice.

Example 95: in vitro characterization of the activity of TGFRt15-TGFRs and TGFRt15-TGFRs

TGFRII was demonstrated to interact with TGF-beta 1-3. However, there is no report in the literature demonstrating the interaction between TGFRII and latent tgfβ. To assess whether TGFRt15-TGFRs, TGFRt15 x-TGFRs and TGFRII-Fc interacted with latent tgfβ, we applied 2.5nM human latent tgfβ1-his tags (cat#tg1-H524 x, acro Biosystems) or control protein CD39-his tags (lot#58-49/51, HCW Biologics)) coated ELISA plates (cat#80040le 0910, zemerfei) overnight at 4 ℃ in 50mM carbonate buffer pH 9.4 (100 μl/well). The next day, ELISA wash buffer (phosphate buffer with 0.05% Tween 20) was used to wash the plates three times, blocking with blocking buffer (1% BSA-PBS) for 1 hour, then decreasing the concentration of TGFRt15-TGFRs, TGFRt15 x-TGFRs, or TGFRII-Fc in blocking buffer from 200nM to 0.09nM, adding to the plates, and incubating the plates at 25℃for 1 hour. The plates were washed three times with ELISA wash buffer. Detection antibody, biotinylated anti-TGFRII antibody (cat#baf241, additively) at 0.1 μg/mL was added to the plate and incubated for 1 hour at 25 ℃. The plates were washed and 0.25 μg/mL horseradish peroxidase-streptavidin (code #016-030-084, jackson immunoresearch laboratory) was added to the plates and incubated for 30 minutes at 25 ℃. The plates were washed and HRP substrate ABTS (cat#abts-1000-01, surmod) was added to the plates and incubated for 20 minutes at 25 ℃. Plates were read with an enzyme-labeled instrument (Multiscan Sky, thermo Scientific) at OD405 nm. As shown in figure 222A, both TGFRt15-TGFRs and TGFRt15-TGFRs interact similarly with latent tgfβ1. However, TGFRII-Fc interacted with latent tgfβ1 with lower affinity than TGFRt15 x-TGFRs (fig. 222B). The results indicate that TGFRt15-TGFRs, TGFRt15 x-TGFRs and TGFRII-Fc can interact with latent tgfβ1, TGFRt15-TGFRs, TGFRt15 x-TGFRs surprisingly show higher affinity interactions than TGFRII-Fc.

Example 96: prothrombin time assay

Prothrombin Time (PT) measurement is intended to measure the time required for coagulation after plasma is mixed with tissue factor and optimal calcium concentration. A mixture of tissue factor and phospholipid (called prothrombin) acts as an enzyme that converts prothrombin to thrombin, which in turn causes blood clotting by converting fibrinogen to fibrin. Since nomovin (inovin) is a lipidated recombinant human TF243, it was used as a standard in our experiments. In PT analysis, shorter PT times (clotting times) indicate higher TF-dependent clotting activity, while longer PT (clotting times) indicate lower TF-dependent clotting activity.

Briefly, 0.1mL of normal human plasma (Ci-Trol coagulation control, grade I) was pre-warmed at 37℃for 3 min. By adding 0.2mL of various diluted inovin (inovin) or in PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6mM CaCl) ₂ 0.1% bsa) was diluted in the test sample (TGFRt 15-TGFRs) to initiate the plasma clotting reaction. Clotting times were monitored and reported by a STart PT analyzer (Sitah diagnostics Inc. (Diagnostics Stago), parsippany, N.J.).

As shown in fig. 223, different amounts of minoxidil (innovin) added to the PT assay (minoxidil reconstituted with purified water equivalent to 10nM of lipidated recombinant human TF243 was regarded as 100% minoxidil) did demonstrate the inverse relationship between the amount of TF243 added to the PT assay and PT time. For example, the PT time of 1% of the inovin is about 25.0 seconds, while the PT time of 100% of the inovin is 8.5 seconds.

FIG. 224 shows the PT test results for TGFRt 15-TGFRs. TGFRt15-TGFRs show prolonged PT time compared to inovin (inovin), almost the same as buffer, indicating very low or no clotting activity.

The clotting effects of TGFRt15-TGFRs in the presence of CTLL cells were also assessed. Binding experiments performed confirm that TGFRt15-TGFRs can bind CTLL cells. The TGFRt15-TGFRs clotting assay in the presence of CTLL cells will more closely reflect the effective clotting activity in vivo. TGFRt15-TGFRs were pre-incubated with CTLL cells in PT assay buffer for 20-30 min at 37 ℃. We then performed PT assays as described above. FIG. 224 shows that the mixture of TGFRt15-TGFRs and CTLL cells has a shorter clotting time (154.6 seconds) than either TGFRt15-TGFRs alone (167.6 seconds) or CTLL cells alone (161.9 seconds). However, the clotting time of 154.6 seconds is still significantly longer than the clotting time of 8.5 seconds for inovin.

In summary, TGFRt15-TGFRs have very low or no TF-dependent clotting activity (i.e. within the physiological range of clotting factors in human plasma), even if cells are present that are capable of binding TGFRt 15-TGFRs.

Example 97: young mice and mice treated with TGFRt15-TGFRs or PBS for short term (10 days) or long term (60 days) Tian (heaven)

Gene expression of senescence markers in tissues of aged mice following follow-up of (a)

C57BL/6, 72 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups and treated subcutaneously with PBS (PBS control group) or 3mg/kg dose of TGFRt15-TGFRs (TGFRt 15-TGFRs group). On day 10 or 60 post-treatment, mice were euthanized and kidneys were harvested and expression levels of the senescence markers PAI1, IL-1 a, IL6 and tnfa were assessed by quantitative PCR. The harvested kidneys were stored in liquid nitrogen in a 1.7mL Eppendorf tube. Samples were homogenized in 1mL of Trizol (zemoeimer) by using a homogenizer. The homogenized tissue was transferred to fresh Eppendorf tubes. Total RNA was extracted using the RNeasy Mini kit (Kaiji # 74106) according to the manufacturer's instructions. 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Kaiji). Using FAM-labeled pre-designed primers from Thermo Scientific, the detection system was used with CFX96 (Bio-Ra d) Real-time PCR was performed. Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct target-Ct 18S. Untreated 6-week-old mice (young mice) were used as controls to compare the gene expression levels of the old mice.

As shown in FIG. 225, the results indicate that the gene expression of PAI-1, IL-1. Alpha., IL-6 and IL-1. Beta. In the kidney increased with increasing age of mice, as expected to increase in the age-dependence of cellular senescence. Treatment of mice 72 months old with a single dose of TGFRt15-TGFRs produced a significant and durable effect in reducing gene expression of senescence markers in the kidneys, indicating that the treatment was associated with a reduction in naturally occurring senescent cells in the kidneys of aged mice.

As shown in fig. 226, the results demonstrate that treatment of mice 72 months old with a single dose of TGFRt15-TGFRs mediated a significant and durable effect in reducing IL-1 a and IL6 gene expression in the liver, indicating that the treatment was associated with a reduction in naturally occurring senescent cells in the liver of old mice.

C57BL/6, 72 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups and treated subcutaneously with PBS (PBS control group) or 3mg/kg dose of TGFRt15-TGFRs (TGFRt 15-TGFRs group). On either day 10 or day 60 post-treatment, mice were euthanized and kidneys were harvested to assess protein levels of the senescence marker PAI-1 by tissue ELISA. The harvested kidneys were stored in liquid nitrogen in a 1.7mL Eppendorf tube. Samples were homogenized in 0.3mL of extraction buffer (Ai Bokang) by using a homogenizer. The homogenized tissue was transferred to fresh Eppendorf tubes. Protein levels in homogenized tissues were quantified using BCA protein assay kit (Pierce). Mouse PAI-1ELISA (Andi organism (R & DSsystem)) was performed using 200mg of tissue homogenate. Based on the standard curve, the concentration of PAI-1 was calculated in picograms per milligram of tissue.

As shown in FIG. 227, protein levels of the aging marker PAI-1 were reduced in kidney of aged mice treated with TGFRt15-TGFRs compared to PBS group at 60 days after treatment. These results are consistent with the effect of TGFRt15-TGFRs treatment on PAI-1 gene expression in the kidneys of aged mice. Taken together, these results demonstrate that a single treatment of TGFRt15-TGFRs produces a significant and durable effect in reducing naturally occurring senescent cells in aged mouse tissues (measured by reduced gene and protein expression of the senescence markers).

Example 98: TGFRt15-TGFRs and TGFRt15-TGFRs (IL-15 mutant) treatment in reduced senior mice Comparison of Gene expression of senescence markers in tissues

C57BL/6, 72 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into the following five groups: saline control group (n=8); TGFRt15-TGFRs group (n=8); IL15S group (n=8); TGFRt15-TGFRs group (n=8); IL15sa+tgfrt15—tgfrs group (n=8). Mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg), IL15SA (0.5 mg/kg) or TGFRt15-TGFRs (3 mg/kg) plus IL15SA (0.5 mg/kg). Mouse blood was prepared to assess the change in different immune cell subsets following treatment with TGFRt15-TGFRs and other agents. On day 17 post-treatment, mouse blood was collected from the submaxillary vein into EDTA-containing tubes. Whole blood was centrifuged in a microcentrifuge at 3000RPM for 10 minutes to collect plasma. Plasma was stored at-80 ℃, while whole blood was subjected to immunocytophenotype analysis by flow cytometry. Whole blood RBCs were lysed in ACK buffer for 5 min at room temperature. The remaining cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To assess different types of immune cells in blood, cells were stained with CD3, CD45, CD8 and NK1.1 antibodies (hundred-advanced biotechnology) that specifically bound to the cell surface for 30 minutes at Room Temperature (RT). After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in FIG. 228, the results demonstrate that aged mice were treated with TGFRt 15-TGFRs. IL15SA (positive control) or TGFRt 15. Times. -TGFRs+IL15SA mediates CD3 in blood ⁺ CD8 ⁺ 、CD3 ^- NK1.1 ⁺ And CD3 ⁺ CD45 ⁺ The percentage of immune cells increased, while treatment with TGFRt15 x-TGFRs had little or no effect on the percentage of these cell populations. These results indicate that IL-15 activity of TGFRt15-TGFRs increases CD8 in blood of aged mice ⁺ T cells and NK cells play a role.

As shown in FIG. 229, the results demonstrate that aged mice were treated with TGFRt 15-TGFRs. IL15SA (positive control) or TGFRt 15. Times. -TGFRs+IL15SA mediates CD3 in spleen ⁺ CD8 ⁺ 、CD3 ^- NK1.1 ⁺ And CD3 ⁺ CD45 ⁺ The percentage of immune cells increased, while treatment with TGFRt15 x-TGFRs had little or no effect on the percentage of these cell populations. These results indicate that IL-15 activity of TGFRt15-TGFRs increases CD8 in the spleen of aged mice ⁺ T cells and NK cells play a role.

C57BL/6, 72 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into the following five groups: saline control group (n=8); TGFRt15-TGFRs group (n=8); IL15S group (n=8); TGFRt15-TGFRs group (n=8); and IL15SA and TGFRt15-TGFRs group (n=8). Mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg), IL15SA (0.5 mg/kg) or TGFRt15-TGFRs (3 mg/kg) plus IL15SA (0.5 mg/kg). The kidney, liver and lung of mice were harvested to assess gene expression of senescence markers p21, PAI1, IL-1 alpha and IL6 by quantitative PCR in tissues or control groups treated with TGFRt15-TGFRs, TGFRt 15-TGFRs. Mice were euthanized on day 17 post-treatment, kidneys, liver and lungs were harvested and stored in liquid nitrogen in 1.7mL Eppendorf tubes. Samples were homogenized in 1mL of Trizol (zemoeimer) by using a homogenizer. The homogenized tissue was transferred to fresh Eppendorf tubes. Total RNA was extracted using the RNeasy Mini kit (Kaiji # 74106) according to the manufacturer's instructions. Using the quanditect reverse transcription kit (qijie ) 1. Mu.g of total RNA was used for cDNA synthesis. Real-time PCR was performed with a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers from Thermo Scientific. Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct target-Ct 18S.

As shown in figures 230A-D, treatment of mice 72 months old with a single dose of TGFRt15-TGFRs or TGFRt15-TGFRs mediated a significant and sustained effect in reducing p21, PAI1, IL-1 a and IL6 gene expression in the kidney and liver, indicating that the treatment was associated with a reduction in naturally occurring senescent cells in the kidney and liver of old mice. The results of this study demonstrate that IL-15 and TGF-beta trap activity of TGFRt15-TGFRs can reduce naturally occurring senescent cells in aged mouse tissues.

Example 99: immunophenotype after IL-15 based agent treatment

Mouse blood was prepared to assess the change in different immune cell subsets following treatment with IL-15 based agents: TGFRt15-TGFRs, IL-15 superagonists (IL-15 SA) and fusion of IL-15 with D8N mutants that knocked out IL-15 activity (TGFRt 15 x-TGFRs). C57BL/6,6 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into groups (n=6/group) and treated with the following method: 1) PBS (saline) control, 2) docetaxel, 3) docetaxel and TGFRt15-TGFRs, 4) docetaxel and IL15SA, 5) docetaxel and IL-15 mutant (TGFRt 15 x-TGFRs) and 6) docetaxel and IL-15 super-agonist (IL-15 SA) plus TGFRt15 x-TGFRs. Three doses of docetaxel (10 mg/kg) were used to induce aging in mice on day 1, day 4 and day 7. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, IL-15SA or a combination of the above. TGFRt15-TGFRs and TGFRt15-TGFRs are administered at a dose of 3mg/kg, whereas IL-15SA is administered at a dose of 0.05 mg/kg. On day 3 after study drug treatment, mouse blood was collected from the submandibular vein into EDTA tubes. Whole blood was centrifuged in a microcentrifuge at 3000RPM for 10 minutes to collect plasma. Plasma was stored at-80 ℃, while whole blood was subjected to immunocytophenotype analysis by flow cytometry. RBCs were lysed in ACK buffer for 5 min at 37 ℃. The remaining cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To assess different types of immune cells in blood, cells were stained with antibodies to CD4, CD45, CD19, CD8 and NK1.1 (hundred-in biotechnology) at Room Temperature (RT) for 30 minutes. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. Cells were treated with permeabilization buffer (invitrogen life technology) for 20 min at 40 ℃ and then washed with permeabilization buffer (invitrogen life technology). Cells were then stained for intracellular proliferation markers (Ki 67) for 30 min at RT. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in fig. 231A and 231B, the results demonstrate that treatment of mice with TGFRt15-TGFRs, IL15SA (positive control) or TGFRt15 x-tgfrs+il 15SA mediated CD8 in blood ⁺ T cells and NK1.1 ⁺ The percentage of cells and the increase in proliferation (measured by Ki 67) while treatment with TGFRt15 x-TGFRs had little or no effect on the percentage of these cell populations. These results indicate that IL-15 activity of TGFRt15-TGFRs is CD8 in blood of aged mice after chemotherapy ⁺ T cells and NK cells play a role.

Example 100: chemotherapy and post-treatment with IL-15 based agents for aging markers in lung and liver tissues of mice Assessment of gene expression of the markers p21 and CD26

After chemotherapy and administration of study treatments, gene expression of cellular senescence markers was assessed in tissues of normal mice. C57BL/6,6 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into six groups and treated as follows: 1) PBS (saline) control (n=5), 2) docetaxel (n=8) 3) docetaxel and TGFRt15-TGFRs (n=8), 4) docetaxel and IL-15SA (n=8), 5) docetaxel and IL-15 mutant (TGFRt 15 x-TGFRs) (n=8), and 6) docetaxel and IL-15 super-agonist (IL-15 SA) plus TGFRt15 x-TGFRs (n=6). Three doses of docetaxel (10 mg/kg) were used to induce aging in mice on day 1, day 4 and day 7. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, IL-15SA or a combination of the following. TGFRt15-TGFRs and TGFRt15-TGFRs are administered at a dose of 3mg/kg, and IL-15SA is administered at a dose of 0.5mg/kg. Mouse tissues were prepared to assess gene expression of different senescence markers. Mice were euthanized on day 7 after study drug treatment, liver and lung tissue was collected and stored in liquid nitrogen in 1.7mL Eppendorf tubes. Samples were homogenized in liquid nitrogen by using a mortar and pestle. The homogenized tissue was transferred to fresh Eppendorf tubes containing 1mL of Trizol (zemoeimer). Total RNA was extracted using the RNeasy Mini kit (QIAGER# 74106) according to the manufacturer's instructions and 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (QIAGER). Real-time PCR was performed with a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers from Thermo Scientific. Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct target-Ct 18S.

As shown in fig. 232, gene expression of senescence markers p21 and CD26 in lung (fig. 232A) and (fig. 232B) and senescence marker p21 in liver (fig. 232C) tissues was reduced in docetaxel-treated mice compared to gene expression in saline-treated mice tissues. The gene expression of senescence markers p21 and CD26 in the lung and in liver was reduced in chemotherapy treated mice following the subsequent combination treatment with TGFRt15-TGFRs, IL-15SA and TGFRt15 x-TGFRs mutants compared to the chemotherapy treated controls. However, TGFRt15 x-TGFRs mutant treatment failed to affect chemotherapy-induced senescence marker gene expression in these tissues. These results indicate that IL-15 activity is important for the clearance of TIS senescent cells in normal tissues of mice.

Example 101: TGFRt15-TGFRs treatment enhances immune cell proliferation in peripheral blood of B16F10 tumor-bearing mice, Amplification and activation

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with anti-TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Tumor-bearing mice treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. Blood was drawn from the submandibular vein on days 3, 5 and 10 following the immunotherapy treatment (day 8). RBCs were lysed in ACK lysis buffer, then lymphocytes were washed and stained with antibodies specific for NK, CD8, CD25 and granzyme B (GzB) (hundred-in biotechnology) cell surface expression for 30 min at Room Temperature (RT). After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at RT. After two washes, the cells were resuspended in fixation buffer. After fixation, cells were washed and treated with permeabilization buffer (invitrogen life technology) for 20 min at 4 ℃ and then washed with permeabilization buffer (invitrogen life technology). Cells were then stained for intracellular proliferation markers (Ki 67) for 30 min at RT. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in figures 233A and B, peripheral blood analysis showed proliferative Ki67 positive NK and CD8 compared to saline or chemotherapy treated groups ⁺ Cells appear mainly on day 3 after TGFRt15-tgfrs+ta99 treatment. NK and CD8 discovery on day 3 and day 5 post immunotherapy ⁺ Expansion of cells. Although NK cells were still expanding, CD8 was not found on day 10 after immunotherapy ⁺ Cells expand in blood. Immune cells in combination with saline or chemotherapy treatmentIn contrast, these cells also expressed the activation markers CD25 and granzyme B after TGFRt15-TGFRs+TA99 treatment. These effects are consistent with the immunostimulatory activity of TGFRt 15-TGFRs.

Example 102: TGFRt15-TGFRs treatment reduced TGF beta levels in plasma of B16F10 tumor bearing mice

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Tumor-bearing mice treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. On days 1, 3, 5 and 10 after the immunotherapy treatment, blood was collected from under the jaw in EDTA-containing tubes and immediately placed on ice. Blood was centrifuged at 3,000rpm for 15 minutes at room temperature to separate plasma. Plasma samples were aliquoted and stored at-80 ℃. Plasma TGF beta levels were analyzed by using the cytokine array TGF beta 3-plex (TGF beta 1-3) from Eve Technologies, calgari, alberta, canada.

As shown in figure 234, the results demonstrate that the administration of TGFRt15-tgfrs+ta99 resulted in reduced levels of TGF- β1, TGF- β2 and TGF- β3 in the plasma of tumor bearing mice, 3 to 5 days after treatment, as compared to saline or chemotherapy-treated groups. This effect is consistent with the TGF-beta agonistic activity of TGFRt 15-TGFRs.

Example 103: TGFRt15-TGFRs treatment reduced pro-inflammatory cytokines in plasma of B16F10 tumor-bearing mice Horizontal level

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with anti-TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Lotus treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7Tumor mice served as controls. Blood was drawn from the submandibular vein on days 1, 3, 5 and 10 after immunotherapy treatment (day 8) into EDTA-containing tubes and immediately placed on ice. Blood was centrifuged at 3,000rpm for 15 minutes at room temperature to separate plasma. Plasma samples were aliquoted and stored at-80 ℃. Aliquots were diluted 2-fold in PBS and analyzed using the mouse cytokine antibody chip pro-inflammatory focusing 10-fold (Mouse Cytokine Array Proinflammatory Focused-plex) (MDF 10) assay.

As shown in figure 235, the results demonstrate that administration of TGFRt15-tgfrs+ta99 reduced plasma levels of IL2, IL-1 beta, IL6, MCP-1 and GM-CSF in tumor bearing mice compared to chemotherapy-treated groups at day 10 post-treatment. This effect is consistent with the immunostimulatory activity of TGFRt 15-TGFRs.

⁺ Example 104: TGFRt15-TGFRs treatment enhances NK and CD8 amplification in spleen of B16F10 tumor bearing mice

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with anti-TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Tumor-bearing mice treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. Mice were sacrificed and spleens were collected on days 3, 5 and 10 following the immunotherapy treatment (day 8). The spleen was crushed with the flat rear end of the sterile piston/plunger of a 3cc syringe to release the spleen cells. Spleen cells were passed through a 70- μm cell filter and homogenized into single cell suspensions. RBCs were lysed in ACK lysis buffer, splenocytes were washed and stained with NK and CD8 (hundred-advanced biotechnology) cell surface expressed antibodies for 30 min at RT. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in FIG. 236, NK and CD8 were observed in the spleen on days 3 and 5 after TGFRt15-TGFRs+TA99 treatment compared to saline or chemotherapy treated group ⁺ CellsIs amplified by (a) and (b). NK cells (but not CD 8) in the spleen of tumor-bearing mice on day 10 post-immunotherapy treatment compared to spleen levels in the chemotherapy-treated group ⁺ Cells) levels are still elevated. These effects are consistent with the immunostimulatory activity of TGFRt 15-TGFRs.

Example 105: TGFRt15-TGFRs treatment enhances glycolytic activity of spleen cells in B16F10 tumor-bearing mice

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with anti-TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Tumor-bearing mice treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. Mice were sacrificed and spleens were collected on days 3, 5 and 10 following the immunotherapy treatment (day 8). The spleen was crushed with the flat rear end of the sterile piston/plunger of a 3cc syringe to release the spleen cells. Spleen cells were passed through a 70- μm cell filter and homogenized into single cell suspensions. RBCs were lysed in ACK lysis buffer, splenocytes were washed and counted. To measure glycolytic activity of spleen cells, cells were washed and resuspended in hippocampal medium (seahorse media) at 4x10 ⁶ Individual cells/mL were resuspended. Cells were seeded at 50 μl/well in Cell-Tak coated hippocampal bioanalyzer (Seahorse Bioanalyzer) XFe96 plates in Seahorse XF RPMI medium supplemented with 2mM L-glutamine, pH 7.4 for glycolytic pressure testing. Cells were attached to the plates at 37 ℃ for 30 minutes. In addition, 130 μl of assay medium was added to each well of the plate (also the background well). The plate was free of CO at 37deg.C ₂ Incubate in incubator for 1 hour. For glycolytic pressure testing, the calibration plate contained a 10x glucose/oligomycin/2 DG solution prepared in a hippocampal (Seahorse) assay medium, and 20 μl of glucose/oligomycin/2 DG was added to each port of the overnight calibrated extracellular flux plate. Glycolytic stress test is based on extracellular acidification rate (ECAR), measuring three key parameters of glycolytic function, includingGlycolysis, glycolytic capacity, and glycolytic reserves. The complete ECAR analysis includes four phases: non-glycolytic acidification (no drug), glycolysis (10 mM glucose), maximum glycolysis induction/glycolysis capacity (2. Mu.M oligomycin), and glycolysis reserves (100 mM 2-DG). At the end of the experiment, the data was exported as a Graph Pad Prism file. The XF glycolysis pressure test report generator automatically calculates XF cell glycolysis pressure test parameters according to Wave data. Data were analyzed using Wave software (Agilent).

As shown in figures 237A and B, splenocytes isolated from tumor-bearing mice on days 3 and 5 after TGFRt15-tgfrs+ta99 treatment showed enhanced basal glycolysis, capacity and reserve compared to splenocytes of saline or chemotherapy-treated groups. However, no significant differences in spleen cell glycolytic activity were observed at day 10 post-immunotherapy. These effects are consistent with the immunostimulatory activity of TGFRt 15-TGFRs.

Example 106: TGFRt15-TGFRs treatment enhances mitochondrial respiration of spleen cells in B16F10 tumor-bearing mice

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with anti-TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Tumor-bearing mice treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. Mice were sacrificed and spleens were collected on days 3, 5 and 10 following the immunotherapy treatment (day 8). The spleen was crushed with the flat rear end of the sterile piston/plunger of a 3cc syringe to release the spleen cells. Spleen cells were passed through a 70 μm cell filter and homogenized to a single cell suspension. RBCs were lysed in ACK lysis buffer, splenocytes were washed and counted. To measure mitochondrial respiration of spleen cells, cells were washed and resuspended in hippocampal medium (seahorse media) and expressed at 4x10 ⁶ Individual cells/mL were resuspended. Cells were seeded at 50 μl/well in Cell-Tak coated hippocampal bioanalyzer (Seahorse Bioanalyzer) XFe96 plates in Seahorse XF RPMI mediumThe medium was supplemented with 2mM L-glutamine, pH 7.4, for glycolytic stress testing. For mitochondrial pressure testing, cells were inoculated in SeaHorse XF RPMI medium supplemented with 10mM glucose and 2mM L-glutamine, pH 7.4. Cells were attached to the plates at 37 ℃ for 30 minutes. In addition, 130 μl of assay medium was added to each well of the plate (also the background well). The plate was free of CO at 37deg.C ₂ Incubate in incubator for 1 hour. For mitochondrial pressure testing, the calibration plate contained a 10x oligomycin/FCCP/rotenone solution prepared in a hippocampal (Seahorse) assay medium, and 20 μl of oligomycin, FCCP and rotenone were added to each port of the overnight calibrated extracellular flux plate. Oxygen Consumption Rate (OCR) was measured using XFe96 extracellular flux analyzer. Complete OCR analysis includes four phases: basal respiration (no drug), ATP-related respiration/proton leakage (1.5 μm mM oligomycin), maximum oxygen consumption (maximal respiration) (2 μm FCCP), and respiratory potential (spare respiration) (0.5 μm rotenone). At the end of the experiment, the data was exported as a Graph Pad Prism file. The XF mitochondrial pressure test report generator automatically calculates XF mitochondrial pressure test parameters from Wave data that has been exported to Excel. Data were analyzed using Wave software (agilent).

As shown in figures 238A and B, splenocytes isolated from tumor-bearing mice on days 3 and 5 after TGFRt15-tgfrs+ta99 therapy showed enhanced basal respiration, mitochondrial respiration, capacity and ATP production compared to splenocytes of saline or chemotherapy-treated groups. However, no significant difference in mitochondrial respiration of spleen cells was observed at day 10 after immunotherapy. These effects are consistent with the immunostimulatory activity of TGFRt 15-TGFRs. It is well known that metabolic pathways such as oxidative metabolism and glycolysis preferentially fuel cell fate decisions and effector functions of immune cells. Thus, TGFRt 15-TGFRs-mediated glycolytic activity and increased mitochondrial respiration might be associated with NK and CD8 in mouse blood, spleen and tumors ⁺ Activation of immune cells.

Example 107: TGFRt15-TGFRs treatment enhances NK and CD8 immune cell infiltration (TILs) into the B16F10 charge Tumor of tumor mice

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with anti-TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Tumor-bearing mice treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. Mice were sacrificed and tumors were harvested on day 3, day 5, and day 10 after immunotherapy treatment. Tumor tissue was dissociated into single cell suspensions by collagenase digestion to determine tumor-infiltrated immune cells. Single cell suspensions were plated on Ficoll-Paque medium and then density gradient centrifuged to separate lymphocytes and tumor cells. The cells were centrifuged at 1000g for 20 min at 20℃while slowly accelerating and then disconnected. After centrifugation, ficoll-Paque resulted in a significant separation between the two layers. TILs were found to be present at the interface between the medium and Ficoll-Paque, whereas the pellet consisted of tumor cells. The TILs were carefully removed from the interface and washed with complete RPMI medium. After washing, RBCs were lysed in ACK buffer for 5 min at room temperature. Cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To evaluate different types of immune cells in tumors, cells were stained with antibodies to CD8, NK1.1, CD25 and GzB (hundred-in biotechnology) at RT for 30 min. After surface staining, the remaining cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After two washes, the cells were resuspended in fixation buffer. After fixation, cells were washed and treated with permeabilization buffer (invitrogen life technology) for 20 min at 4 ℃ and then washed with permeabilization buffer (invitrogen life technology). Cells were then stained for intracellular proliferation markers (Ki 67) for 30 min at RT. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in figures 239A and B, tumor analysis showed high levels of Ki67 positive NK and CD8 cells at day 3 post-therapy. NK and CD8 were found on days 3 and 5 after TGFRt15-TGFRs+TA99 therapy compared to chemotherapy-treated group ⁺ Expansion of cells (based on the percentage of lymphocytes in the tumor). Tumor CD8 even at day 10 after immunotherapy ⁺ Cells will also rise. NK and CD8 on day 3 after TGFRt15-TGFRs+TA99 therapy compared to immune cells of chemotherapy-treated group ⁺ Both showed expression of the activation markers CD25 and granzyme B. These effects are consistent with the immunostimulatory activity of TGFRt15-TGFRs and are comparable to the changes observed in blood and spleen cells of tumor-bearing mice.

Example 108: histopathological analysis of tumors after TGFRt15-TGFRs treatment

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 and a single dose of TGFRt15-TGFRs (3 mg/kg) on day 8 in combination with anti-TYRP-1 antibody TA99 (tumor antigen-targeted monoclonal antibody) (200 μg). Tumor-bearing mice treated with saline or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. Blood was drawn from the submandibular vein on days 1, 3, 5 and 10 after the immunotherapy treatment (day 8). On day 10 after immunotherapy, mice were sacrificed and tumors were isolated. For histological analysis, tumor samples were fixed in 10% formalin solution, embedded in paraffin and cleaved at 5 μm. H for slicing &E staining to assess tissue and cell morphology. Slides were scored for mitotic and necrotic activity of tumors. Percent tumor necrosis scores were +1 (0-20%), +2 (20-40%) and +3 (40-60%). Tumors have mitotic index scores of +1=medium (1-5 per high power field) and +2=large (extension) (> 5 per high power field).

As shown in figure 240, tumors showed less mitotic and necrotic activity after TGFRt15-tgfrs+ta99 treatment. Mitotic index is related to dividing cells and the presence of necrosis is a measure of more invasive character and poor prognosis. Thus, TGFRt15-TGFRs is a promising therapy in preclinical mouse models for testing combined tumor immunotherapy.

Example 109: anti-PD-L1 antibodies in combination with TGFRt15-TGFRs+TA99 in B16F10 melanoma mouse model And chemotherapy

Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. Following tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7. Tumor-bearing mice treated with saline alone or docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 served as controls. The remaining mice were randomized into two groups, one group treated with anti-mPD-L1 antibody (2X 10 mg/kg) and the other group treated with TGFRt15-TGFRs (3 mg/kg) and TA99 (200. Mu.g) on day 8. After 6 days, mice receiving TGFRt15-TGFRs and TA99 received anti-mPD-L1 antibody (2X 10 mg/kg), and mice receiving anti-mPD-L1 antibody were treated with TGFRt15-TGFRs (3 mg/kg) and TA99 (200. Mu.g). anti-mPD-L1 antibodies were administered in two doses on day 8 and day 10 or day 14 and day 16. Tumor growth was monitored by caliper measurement and tumor volume was calculated using the formula v= (LxW 2)/2, where L is the maximum tumor diameter and W is the perpendicular tumor diameter. N=6-8 mice/group.

As shown in figure 241, in B16F10 tumor bearing mice, the administration of TGFRt15-tgfrs+ta99 followed by the administration of anti-PD-L1 antibody resulted in better anti-tumor activity than the administration of the anti-PD-L1 antibody followed by the TGFRt15-tgfrs+ta99 treatment. Thus, combining TGFRt15-TGFRs with anti-PD-L1 antibodies may be advantageous for the treatment of tumors that are resistant to anti-PD-L1 antibody therapies.

Example 110: the anti-tumor efficacy of TGFRt15-TGFRs in B16F10 melanoma mouse model is NK dependent ⁺ And CD 8T cells

Three doses of NK1.1 Ab (500 μg) or CD8 are used every three days ⁺ a (500 μg) antibody the C57BL/6 mice group (n=6-8 mice/group) was treated intraperitoneally to deplete NK and CD8 cells. Blood was drawn and analyzed for NK and CD8 prior to B16F10 tumor implantation ⁺ LymphocytesHorizontal. Untreated mice served as immunocompetent controls. Subcutaneous injection of 0.5x10 to C57BL/6 mice ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of docetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7 followed by a single dose of TGFRt15-TGFRs (3 mg/kg) +ta99 (200 μg) on day 8. Tumor growth was monitored by caliper measurement and tumor volume was calculated using the formula v= (l×w2)/2, where L is the maximum tumor diameter and W is the perpendicular tumor diameter.

As shown in figure 242, B16F10 tumor-bearing mice treated with TGFRt15-TGFRs in combination with TA99 and chemotherapy showed a significant decrease in B16F10 tumor volume compared to tumors of either saline or chemotherapy-treated groups. However, when NK and CD8 in mice ⁺ When cell subsets were depleted, immunotherapy had no effect on antitumor activity. This experiment shows that NK and CD8 ⁺ Immune cells play an important role in TGFRt15-TGFRs mediated antitumor activity.

Example 111: TGFRt15-TGFRs and TGFRt15-TGFRs treatment reduced B16F10 tumor burden following chemotherapy Comparison of senescence markers in liver and lung tissues of mice

C57BL/6,6-8 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were divided into the following five groups: saline control group (n=7), docetaxel (DTX) group (n=7), dtx+tgfrt15-TGFRs group (n=7), dtx+il15SA group (n=7). B16F10 tumor cells (1 x10 on day 0 ⁷ Individual cells/mice) were implanted into mice. Mice were treated subcutaneously with 10mg/kg docetaxel on days 1, 4 and 7. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg) or IL15SA (0.5 mg/kg). Mice were euthanized on day 17 post-treatment and liver and lung were harvested in order to assess gene expression of senescence markers p21, IL-1 alpha and IL6 in liver and senescence markers p21 and IL-1 alpha in lung by quantitative PCR in tissues treated with TGFRt15-TGFRs or TGFRt15 x-TGFRs and control group. The harvested organ was stored in liquid nitrogen in a 1.7mL Eppendorf tube. By using The homogenizer homogenizes the sample in 1mL of Trizol (zemoeimeric). The homogenized tissue was transferred to fresh Eppendorf tubes. Total RNA was extracted using the RNeasy Mini kit (Kaiji # 74106) according to the manufacturer's instructions. 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Kaiji). Real-time PCR was performed with a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers from Thermo Scientific. Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct target-Ct 18S.

As shown in fig. 243, gene expression of senescence markers p21, IL-1 a and IL6 in liver (a) and lung (B) tissues was shown to be reduced in both TGFRt15-TGFRs and TGFRt15-TGFRs treated tumor-bearing mice when compared to gene expression in tissues of chemotherapy treated mice.

Example 112: TGFRt15-TGFRs treatment reduces chemotherapy-induced senescent tumor cells in vivo

B16F10 melanoma cells were transduced stably with GFP lentiviral plasmids and GFP-expressing tumor cells (B16F 10-GFP) were selected by growth in puromycin-containing medium. As analyzed by FACS, almost 95% of B16F10 melanoma cells were GFP positive. To induce senescence, B16F10-GFP cells were treated with 7.5. Mu.M Docetaxel (DTX) for 3 days and then recovered in normal growth medium for 4 days. To quantify gene expression of senescence markers and NK cell ligands, docetaxel-treated B16F10 GFP cells (B16F 10-GFP-SNC) were homogenized in 1mL of Trizol (Sieimer's fly) using a homogenizer. The homogenized cells were transferred to fresh Eppendorf tubes. Total RNA was extracted using the RNeasy Mini kit (Kaiji # 74106) according to the manufacturer's instructions. 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Kaiji). Real-time PCR was performed with a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers from Thermo Scientific. Reactions were performed in triplicate for all genes tested. Will manage the house base Since 18S ribosomal RNA was used as an internal control to normalize variability in expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct target-Ct 18S. The expression of the different genes in B16F10-GFP-SNC cells was plotted as fold change compared to untreated B16F10-GFP cells.

As shown in FIG. 244, real-time PCR analysis showed that docetaxel in vitro treated B16F10-GFP cells up-regulated gene expression of senescence markers (p 21, H2AX and IL 6) and NK cell ligands (Rae-1 e and ULBP-1) compared to untreated B16F10-GFP cells.

To determine whether chemotherapy-induced senescent tumor cells were reduced by in vivo immunotherapy, B16F10 parental melanoma cells (0.75 x10 ⁶ And B16F10-GFP-SNC cells (0.75 x 10) ⁶ Individual) and subcutaneously injecting the cell mixture into C57BL/6 mice. Mice were also injected with B16F10 and B16F10-GFP cells as controls. The B16F10 parent cells will grow into tumors, while the B16F10-GFP-SNC cells will become part of the tumor microenvironment. When the tumor reaches about 350mm ³ At this time, mice bearing mixed tumors were divided into 2 groups. One group received PBS subcutaneously as a control, and the other group received TGFRt15-TGFRs (3 mg/kg) and TA99 (200. Mu.g) subcutaneously. Mice were sacrificed on day 4 after immunotherapy treatment. Tumor tissue was dissociated into single cell suspensions by collagenase digestion to determine tumor-infiltrated immune cells. Single cell suspensions were plated on Ficoll-Paque medium and then density gradient centrifuged to separate lymphocytes and tumor cells. The cells were centrifuged at 1000g for 20 min at 20℃while slowly accelerating and then disconnected. After centrifugation, ficoll-Paque resulted in a significant separation between the two layers. TILs were found to be present at the interface between the medium and Ficoll-Paque, whereas the pellet consisted of tumor cells. The TILs were carefully removed from the interface and washed with complete RPMI medium. After washing, RBCs were lysed in ACK buffer for 5 min at room temperature. The remaining cells were washed in FACS buffer (1 XPBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To evaluate different types of immune cells in tumors, a test is performed at R Cells were stained with CD3, CD45, CD8 and NK1.1 antibodies (hundred-advanced biotechnology) specifically binding to the cell surface for 30 min at T. After surface staining, cells were washed in FACS buffer (1 XPBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5 minutes at 1500RPM at room temperature. After two washes, the cells were resuspended in fixation buffer. After fixation, cells were washed and treated with permeabilization buffer (invitrogen life technology) for 20 min at 4 ℃ and then washed with permeabilization buffer (invitrogen life technology). Cells were then stained for intracellular proliferation markers (Ki 67) for 30 min at RT. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in figure 245, CD8 in tumors 4 days after TGFRt15-tgfrs+ta99 treatment compared to control ⁺ The percentage of T cells and Natural Killer (NK) cells increases. These results indicate that TGFRt15-TGFRs are able to stimulate CD8 in tumors ⁺ Proliferation of T cells and NK cells. CD8 based on measurement of Ki67 marker ⁺ T cells and NK immune cells are also able to proliferate in tumors.

To determine whether chemotherapy-induced senescent tumor cells were reduced by in vivo immunotherapy, B16F10 parental melanoma cells (0.75 x10 ⁶ And B16F10-GFP-SNC cells (0.75 x 10) ⁶ Individual) and subcutaneously injecting the cell mixture into C57BL/6 mice. Mice were also injected with B16F10 and B16F10-GFP cells as controls. The B16F10 parent cells will grow into tumors, while the B16F10-GFP-SNC cells will become part of the tumor microenvironment. When the tumor reaches about 350mm ³ At this time, mice bearing mixed tumors were divided into 2 groups. One group received PBS subcutaneously as a control, and the other group received TGFRt15-TGFRs (3 mg/kg) and TA99 (200. Mu.g) subcutaneously. Mice were sacrificed on day 4 and day 10 after immunotherapy treatment. Tumor tissue was dissociated into single cell suspensions by collagenase digestion to determine tumor-infiltrating immune cells and GFP-positive cells in the tumor. Flow cytometry analysis of tumor cells (fig. 246A) showed that mice treated with immunotherapy showed 4 days and 10 days after treatment compared to PBS control groupA smaller number of GFP positive cells. Tumor cells were seeded in 24-well plates for evaluation by fluorescence microscopy (fig. 246B).

Microscopic images also showed fewer GFP positive cells in tumors of immunotherapy-treated mice compared to control PBS-treated groups. GFP expression in tumors was associated with chemotherapy-induced B16F10-GFP senescent cells, so a decrease in GFP expression following immunotherapy treatment indicates successful elimination of senescent tumor cells in tumor-bearing mice.

Example 113: determination of renal injury induced by cisplatin and after treatment with TGFRt15-TGFRs by tissue ELISA TGF beta levels in kidneys

Mouse kidneys were harvested to assess protein level changes in the senescence marker tgfβ following cisplatin-induced kidney injury and TGFRt15-TGFRs treatment. C57BL/6,8 week old mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were injected weekly with cisplatin (5 mg/kg, intraperitoneally) for 3 weeks to induce kidney injury. One week after cisplatin, mice were treated with PBS or TGFRt15-TGFRs (3 mg/kg) (n=8/group). Mice were euthanized 30 days after immunotherapy treatment, kidneys were harvested and stored in liquid nitrogen in 1.7mL-Eppendorf tubes. Samples were homogenized in 0.3mL of extraction buffer (Ai Bokang) by using a homogenizer. The homogenized tissue was transferred to fresh Eppendorf tubes. Protein levels in homogenized tissues were quantified using BCA protein assay kit (pierce). Mouse TGF-beta ELISA (Andi organism) was performed in 200. Mu.g of tissue. The concentration of tgfβ was calculated per mg of tissue.

As shown in figure 247, TGFRt15-TGFRs treated mice kidney decreased in tgfβ levels compared to PBS control. These results indicate that TGFRt15-TGFRs treatment can provide sustained activity in reducing tgfβ levels in tissues of chemotherapy mice.

Example 114: toxicity of subcutaneous administration of TGFRt15-TGFRs to mice

To further evaluate the dose-dependent toxicological effects of TGFRt15-TGFRs, female C57BL/6 mice (n=3/group) were subcutaneously administered either one or two consecutive doses (every two weeks) of PBS or 3, 10, 50 and 200mg/kg of TGFRt15-TGFRs. Animals were monitored for signs of study drug-related toxicity, changes in body weight during the study, and hematology and serum chemistry parameters at day 7 post-dosing. Mice receiving 200mg/kg of TGFRt15-TGFRs started to show significant weight loss 4 days after the first injection (study day 0 (SD)) and reached a minimum between SD6-9, then recovered to pre-dose level by SD11 (fig. 248A). At SD9, death of one mouse was observed in the 200mg/kg group. There was no apparent treatment-mediated effect on body weight or other clinical signs in any other dose group or after the second dose of 200mg/kg of TGFRt15-TGFRs. After one or two doses of TGFRt15-TGFRs, spleen weight increased in a dose-dependent manner (fig. 248B). Mice also had 25-fold increase in WBC count 7 days after a single dose of 200mg/kg of TGFRt15-TGFRs compared to PBS group, and mice still remained 5-fold higher in WBC count 7 days after the second dose of 200mg/kg (fig. 248C, tables 3 and 4). WBC subset analysis showed a 16-fold increase in absolute lymphocyte count at SD7 for the 200mg/kg group, with > 50-fold increase in neutrophil, monocyte, eosinophil and basophil counts. Although these changes were not observed at lower dose levels of TGFRt15-TGFRs, they were similar to those reported by C57BL/6 mice treated subcutaneously with IL-15/IL-15Rα complex (Liu et al, cytokine 107:105-112, 2018). Other hematological and serum chemistry parameters were similar in TGFRt15-TGFRs and PBS treated animals and were generally within the expected range for C57BL/6 mice (tables 3 and 4). TGFRt15-TGFRs mediated effects were greatest 7 days after the first dose and decreased after the second dose, consistent with previous studies, indicating that mice had reduced immune responses after repeated administration of IL-15/IL-15Rα (Elpek et al, PNAS 107:21647-21652, 2010; frutoso et al, J Immunol 201:493-506, 2018). Overall, C57BL/6 mice were well tolerated by TGFRt15-TGFRs at dose levels up to 50 mg/kg.

TABLE 3 hematology and serum chemistry parameters of C57BL/6 mice on study day 7 after a single dose of TGFRt 15-TGFRs.

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Table 4 hematology and serum chemistry parameters of C57BL/6 mice on study day 21 after two doses of TGFRt 15-TGFRs.

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^a One of the three mice in the TGFRt15-TGFRs group at 50mg/kg observed an AST value of 1586U/L (-6 xULN). This mouse showed no clinical symptoms and its ALT value (61U/L) was in the normal range.

Example 115: chelation of TGF-beta by TGFRt15-TGFRs and TGFRt15-TGFRs in mice

Female C57BL/6 mice were subcutaneously injected with PBS or 3mg/kg of TGFRt15-TGFRs or TGFRt15-TGFRs and plasma was collected at various times after treatment. TGF-beta 1 and TGF-beta 2 plasma levels were determined using the TGF-beta 3-Plex assay (Eve Technologies, canada, alberta, calgari). TGFRt15-TGFRs and TGFRt15-TGFRs were found to significantly reduce plasma TGF- β1 and TGF- β2 levels in C57BL/6 mice 2 days post-treatment (figure 249), consistent with the activity of the tgfbrii domains of these fusion proteins.

Example 116: effects of TGFRt15-TGFRs and TGFRt15-TGFRs on immune cell metabolism in vivo and in vitro

To assess the effect of treatment mediated on immune cell metabolism, extracellular flux assays were performed on splenocytes isolated from mice 4 days after administration of PBS, TGFRt15-TGFRs, or IL-15/IL-15R (IL 15 SA). Extracellular flux analysis was performed on mouse spleen cells using an XFp analyzer (Seahorse Bioscience). As expected, TGFRt15-TGFRs and IL-15 increased the rate of glycolytic capacity (ECAR) (fig. 250A) and mitochondrial respiratory capacity (OCR) (fig. 250B) of isolated splenocytes in a dose level dependent manner. In vivo TGFRt15-TGFRs treatment also increased ECAR and OCR of spleen cells. This was not observed when splenocytes from untreated C57BL/6 mice were incubated with TGFRt15 x-TGFRs for 4 days in vitro. Only TGFRt15-TGFRs (but not TGFRt 15-TGFRs) were able to increase splenocyte ECAR and OCR in vitro at physiologically relevant concentrations (FIGS. 251A-251B). This suggests that both the IL-15 and TGF-beta RII domains of TGFRt15-TGFRs have the effect of stimulating immune cell metabolism in vivo.

Example 117: TGFRt15-TGFRs and TGFRt15-TGFRs against B16F10 black in C57BL/6 mice Antitumor efficacy of melanoma

To evaluate the antitumor efficacy of TGFRt15-TGFRs and TGFRt15 x-TGFRs, the mouse B16F10 tumor model was selected because it is highly invasive, poorly immunogenic and non-immune infiltrating, expresses TGF- β that plays a role in its growth and is resistant to cytokine and checkpoint blocking immunotherapy. B16F10 melanoma cells (5 x10 ⁵ Individual cells) (CRL-6475, atcc) were subcutaneously injected into C57BL/6 mice, followed by PBS, TGFRt15-TGFRs (3 or 20 mg/kg) or TGFRt15-TGFRs (3 or 20 mg/kg) subcutaneously injected on day 1 and day 4 after tumor implantation. Tumor volume was measured every other day and tumors were sacrificed at 4000mm or more according to IACUC specifications ³ Is a mouse of (2). Mice survival was also assessed throughout the study period. When compared by SD15 (i.e., prior to animal death), treatment with 20mg/kg of TGFRt15-TGFRs or TGFRt15 x-TGFRs resulted in significantly slower tumor growth than observed in PBS-treated mice (figure 252A). Tumor bearing mice treated with 20mg/kg of TGFRt15-TGFRs also showed prolonged survival compared to the 3mg/kg of TGFRt15-TGFRs and PBS treated group (FIG. 252B). These results indicate that TGFRt15-TGFRs and TGFRt15 x-TGFRs have antitumor activity against solid B16F10 melanoma, and the bifunctional TGFRt15-TGFRs complex exhibits greater efficacy. Thus, both TGFβRII and IL-15/IL-15RαSu domains are in TGFRt15-TGFRs mediated targeting B1 Plays a role in the activity of 6F10 tumors.

TGFRt15-TGFRs treatment can significantly increase the number of NK and T cells in vivo. To determine whether these immune cells were responsible for TGFRt15-TGFRs mediated antitumor efficacy, CD8 was performed in tumor-bearing mice prior to TGFRt15-TGFRs treatment ⁺ T cells and NK1.1 ⁺ Antibody immune depletion of cells. Found NK1.1 ⁺ Cell depletion (alone or with CD 8) ⁺ T cell depletion combination) abrogated the anti-tumor effect of TGFRt15-TGFRs in B16F10 tumor bearing mice during the first 2 weeks after treatment (fig. 252C), while NK1.1 ⁺ Cell depletion or CD8 ⁺ T cell depletion reduced the survival benefit seen with TGFRt15-TGFRs (figure 252D). Consistent with these findings, TGFRt15-TGFRs treatment also promoted NK cells and CD8 ⁺ An increase in T cell infiltration into B16F10 tumors (figure 252E). These results support the following conclusions: CD8 ⁺ T cells and NK cells play a major role in TGFRt15-TGFRs mediated activity against C57BL/6 mouse melanoma tumor cells.

Example 118: TGFRt15-TGFRs improved glucose control in db/db mice

Male BKS. Cg-Dock7m+/+ Leprdb/J (db/db) mice (Jackson laboratories) five weeks old were fed standard diet and maintained under standard conditions. At weeks 6 and 12 after the start of the study, mice (n=5/group) received subcutaneous injections of PBS (control group) or TGFRt15-TGFRs (3 mg/kg) (treatment group). Fasting blood glucose and insulin were checked three weeks after dose 1. Fasting blood glucose was significantly reduced after TGFRt15-TFGRs treatment (fig. 253A), but blood insulin levels were not altered (fig. 253B) compared to the control.

Example 119: TGFRt15-TGFRs significantly down-regulates aging index and SASP index

Five week old male BKS. Cg-Dock7 ^m +/+Lepr ^db The J (db/db) mice were fed standard diet and were allowed to drink ad libitum. At six weeks of age, mice were randomly assigned to control and treatment groups (n=5/group). Treatment groups received 3mg/kg of TGFRt15-TGFRs subcutaneously from week 6 and week 12 of the study, while control groups received vehicle only (PBS). At the end of the study (4 weeks after dose 2), mice were sacrificed and pancreas collected. Half of the pancreas was homogenized with TRIzol reagent (invitrogen life technology) and total tissue RNA was purified with RNeasy Mini kit (qiagen). cDNA was synthesized using the QuantiTect reverse transcription kit (Kanji), using SsoAdvance according to the comparative threshold cycle method of the manufacturer's protocol ^TM UniversalQuantitative PCR was performed with Green super mix (BioRad) and Quantistudio 3 real-time fluorescent quantitative PCR System (Applied Biosystems). Amplification reactions were performed in duplicate and fluorescence curves were analyzed using software attached to the QuantiStudio 3 real-time PCR system. Housekeeping gene 18s ribosomal RNA was used as an endogenous control reference. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct _Target(s) –Ct _18S . As shown in FIG. 254A, TGFRt15-TGFRs treatment of db/db mice resulted in reduced gene expression of p16, p21, igfr1 and Bamb1 of the aged gene index of pancreas and IL-1α, IL-6, MCP-1 and TNF α of the SASP gene index, as compared to the control group. In general, gene expression of the SASP index and senescence index of pancreas was significantly reduced after TGFRt15-TGFRs treatment compared to control group, while gene expression of the beta cell index of pancreas was not significantly changed in db/db mice treated with TGFRt15-TGFRs and PBS (FIGS. 254B, 254C, 254D). The data indicate that TGFRt15-TGFRs have potent senolytic (senolytic) and reverse senescent (senomorphic) activity, reducing senescent and SASP factors in db/db mouse pancreas. / >

Example 120: TGFRt15-TGFRs reduce senescent cells of pancreatic beta cells

Five week old male BKS. Cg-Dock7m+/+ Leprdb/J (db/db) mice (Jackson laboratories) were fed standard diet (irradiated 2018Teklad global 18% protein rodent diet, envigo) and allowed to drink ad libitum. At six weeks of age, mice were randomly assigned to control and treatment groups (n=5/group). Treatment groups received 3mg/kg of TGFRt15-TGFRs subcutaneously from week 6 and week 12 of the study, while control groups received vehicle only (PBS). At the end of the study (4 weeks after dose 2), mice were sacrificed and pancreas removed in whole, fixed by immersion in 4% formaldehyde (4% formaldehyde in 0.1M phosphate buffer; PBS pH 7.4) and stored at 4 ℃ until further treatment. The dissected pancreas was subjected to paraffin, embedding and slicing, and three 10mm sections (150 mm apart) were cut from each block, representing a systematically uniform random sample of the whole pancreas for each animal.

Multispectral imaging was performed using a Akoya Vectra Polaris instrument. The instrument allows phenotyping, quantification and spatial relationship analysis of tissue infiltration in formalin fixed paraffin embedded biopsies. To quantify pancreatic insulin ⁺ P21 levels in islet regions, formalin fixed paraffin embedded tissue sections were stained consecutively with specific primary antibodies according to standard protocols provided by Akoya and routinely performed by HIMSR. Briefly, slides were dewaxed, heat treated in antigen retrieval buffer, blocked, and incubated with rabbit primary antibodies to insulin (# 4590, cell signaling technology (Cell Signaling Technology)) and p21 (EPR 362, ai Bokang), followed by incubation with horseradish peroxidase (HRP) -conjugated secondary antibody polymer (anti-rabbit) and HRP-reactive OPAL fluorescent reagents (OPAL-520 for insulin and OPAL-570 for p21, akoya) that deposit dyes on the tissue immediately surrounding each HRP molecule using TSA chemistry. To prevent further deposition of fluorescent dye in the subsequent staining steps, the slides were peeled off between each staining while heat treatment was performed in antigen retrieval buffer (citrate buffer for insulin and EDTA buffer for p 21). Using a Akoya Vectra Polaris instrument, the entire slide scan was collected using a 20x objective with a resolution of 0.5 microns. Analysis of 3 color images using the inForm software (Akoya) to isolate adjacent fluorochromes, subtracting autofluorescence, dissecting tissue insulin ⁺ The region, comparing the frequency and location of the cells, dividing the cytoplasmic and nuclear regions, and infiltrating the cells according to the phenotype of cell marker expression.

As shown in FIGS. 255A-255D, p21 positive senescent cells (OPAL-570) accumulated more in insulin positive islet beta cells (OPAL-520) in the pancreas of the control group (FIG. 255A), while these senescent cells were reduced in the pancreas of the TGFRt15-TGFRs treated group (FIG. 255B). Insulin positive islet cells were significantly increased in the TGFRt15-TGFRs treated group compared to the control group (p=0.0278, fig. 255C). The p21 positive senescent beta cells (insulin positive) were reduced in the TGFRt15-TGFRs treated group compared to the control group, but the differences were not statistically significant (fig. 255D). Overall, the data indicate that TGFR15-TGFRs have senescence activity to eliminate senescent cells and promote restoration of normal functional islet beta cells in the pancreas of db/db mice.

⁺ Example 121: TGFRt15-TGFRs reduce pancreatic beta cell attenuation by increasing NK, NKT and CD 8T cells Old cells

Five week old male BKS. Cg-Dock7m+/+ Leprdb/J (db/db) mice (Jackson laboratories) were fed standard diet (irradiated 2018Teklad global 18% protein rodent diet, envigo) and allowed to drink ad libitum. At six weeks of age, mice were randomly assigned to control and treatment groups (n=5/group). Treatment groups received 3mg/kg of TGFRt15-TGFRs subcutaneously from week 6 and week 12 of the study, while control groups received vehicle only (PBS).

Four days after dose 1 treatment, blood was collected and whole blood cells (50 mL) were treated with ACK (ammonium-chloride-potassium) lysis buffer to lyse erythrocytes. Lymphocytes were then stained with PE-Cy 7-anti-CD 3, BV 605-anti-CD 45, perCP-Cy5.5-anti-CD 8a, BV 510-anti-CD 4 and APC-anti-NKp 46 antibodies (all from the hundred-in biotechnology) to assess populations of T cells, NKT cells and NK cells. As shown in FIGS. 256A-256C, CD8 in blood of db/db mice treated with TGFRt15-TGFRs compared to PBS treated mice ⁺ T cells, CD3 ⁺ NKP46 ⁺ NKT cells and CD3 ^- NKP46 ⁺ The percentage of NK cells increases.

Example 122: phenotypic analysis of immune cell subsets in cynomolgus monkey peripheral blood following administration of TGFRt15-TGFRs

On study day 1 and day 15, cynomolgus monkeys (each group) were treated subcutaneously with PBS (vehicle) or with 1, 3 or 10mg/kg of TGFRt15-TGFRs (each group5M: 5F). Blood was collected before treatment (day 1) and on days 5, 22 and 29 after treatment. PBMC were prepared and stained with a panel of fluorescent conjugated antibodies to assess B cells, NK-T cells, treg cells, and CD4 by flow cytometry ⁺ And CD8 ⁺ Phenotype of T cells. FIG. 257 shows that administration of TGFRt15-TGFRs results in Ki67 at day 5 post-treatment ⁺ NK cells, NK-T cells, treg cells and CD4 ⁺ And CD8 ⁺ The percentage of T cells increases significantly. These findings indicate that TGFRt15-TGFRs treatment induces proliferation of these lymphocyte subsets in non-human primates. No effect of treatment on Ki67 expression in B cells was observed.

Example 123: IL-15 immunostimulatory and TGF-beta antagonist activity of TGFRt15-TGFRs

Six week old (young) and 72 week old (old) C57BL/6 mice were subcutaneously injected with a single dose of PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15-TGFRs (3 mg/kg). On day 4 post-treatment, mice were sacrificed and spleens were harvested. The spleen was crushed with the flat rear end of the sterile piston/plunger of a 3cc syringe to release the spleen cells. Spleen cells were passed through a 70 μm cell filter and homogenized to a single cell suspension. RBCs were lysed in ACK lysis buffer, splenocytes were washed and counted. To measure glycolytic activity of spleen cells, cells were washed and resuspended in hippocampal medium (Seahorse media) at 4X10 ⁶ Individual cells/mL were resuspended. Cells were seeded at 50 μl/well in Cell-Tak coated hippocampal bioanalyzer (Seahorse Bioanalyzer) XFe96 plates in Seahorse XF RPMI medium supplemented with 2mM L-glutamine, pH 7.4 for glycolytic pressure testing. Cells were attached to the plates at 37 ℃ for 30 minutes. In addition, 130 μl of assay medium was added to each well of the plate (also the background well). The plate was free of CO at 37deg.C ₂ Incubate in incubator for 1 hour. For glycolytic pressure testing, the calibration plate contained a 10x glucose/oligomycin/2 DG solution prepared in a hippocampal (Seahorse) assay medium, and 20 μl of glucose/oligomycin/2 DG was added to each port of the overnight calibrated extracellular flux plate. Glycolytic stress test based on extracellular acidification rate (ECAR), three measures of glycolytic functionKey parameters include glycolysis, glycolytic capacity and glycolytic reserves. The complete ECAR analysis includes four phases: non-glycolytic acidification (no drug), glycolysis (10 mM glucose), maximum glycolysis induction/glycolysis capacity (2. Mu.M oligomycin), and glycolysis reserves (100 mM 2-DG). At the end of the experiment, the data was exported as a Graph Pad Prism file. The XF glycolysis pressure test report generator automatically calculates XF cell glycolysis pressure test parameters according to Wave data. Data were analyzed using Wave software (Agilent).

As shown in figure 258, splenocytes isolated from aged mice on day 4 after TGFRt15-TGFRs treatment showed enhanced basal glycolysis, glycolysis capacity and glycolysis reserve compared to splenocytes of PBS or TGFRt15-TGFRs treated group. The spleen cells of the aged control mice had lower glycolytic function than the young control mice. Young and old mice treated with TGFRt15-TGFRs were able to increase spleen cell glycolytic function. However, TGFRt15-TGFRs treated old mice were able to increase spleen cell basal glycolytic rate, glycolytic capacity and glycolytic reserves to levels comparable to those observed in TGFRt15-TGFRs treated young mice spleen cells. These findings indicate that IL-15 immunostimulatory and TGF-beta antagonist activity of TGFRt15-TGFRs effectively stimulate and restore the immunocytopenic metabolic activity of aging mice.

Six week old (young) and 72 week old (old) C57BL/6 mice were subcutaneously injected with a single dose of PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15-TGFRs (3 mg/kg). On day 4 post-treatment, mice were sacrificed and spleens were harvested. The spleen was crushed with the flat rear end of the sterile piston/plunger of a 3cc syringe to release the spleen cells. Spleen cells were passed through a 70 μm cell filter and homogenized to a single cell suspension. RBCs were lysed in ACK lysis buffer, splenocytes were washed and counted. To measure mitochondrial respiration of spleen cells, cells were washed and resuspended in hippocampal medium (Seahorse media) and expressed at 4x10 ⁶ Individual cells/mL were resuspended. Cells were seeded at 50 μl/well in Cell-Tak coated hippocampal bioanalyzer (Seahorse Bioanalyzer) XFe96 plates in Seahorse XF RPMI medium supplemented with 2mM L-glutamine, pH 7.4 for glycolytic pressure testing. For wiresGranulocyte stress test cells were inoculated in SeaHorse XF RPMI medium supplemented with 10mM glucose and 2mM L-glutamine, pH 7.4. Cells were attached to the plates at 37 ℃ for 30 minutes. In addition, 130 μl of assay medium was added to each well of the plate (also the background well). The plate was free of CO at 37deg.C ₂ Incubate in incubator for 1 hour. For mitochondrial pressure testing, the calibration plate contained a 10x oligomycin/FCCP/rotenone solution prepared in a hippocampal (Seahorse) assay medium, and 20 μl of oligomycin, FCCP and rotenone were added to each port of the overnight calibrated extracellular flux plate. Oxygen Consumption Rate (OCR) was measured using XFe96 extracellular flux analyzer. Complete OCR analysis includes four phases: basal respiration (no drug), ATP-related respiration/proton leakage (1.5 μm oligomycin), maximum oxygen consumption (2 μm FCCP) and respiratory potential (0.5 μm rotenone). At the end of the experiment, the data was exported as a Graph Pad Prism file. The XF mitochondrial pressure test report generator automatically calculates XF mitochondrial pressure test parameters from Wave data that has been exported to Excel. Data were analyzed using Wave software (agilent).

As shown in figure 259, splenocytes isolated from aged mice on day 4 after TGFRt15-TGFRs therapy showed enhanced basal respiration, ATP-related respiration, maximum oxygen consumption, and reserve capacity (reserve capacity) compared to splenocytes of PBS or TGFRt15-TGFRs treated group. Young and old mice treated with TGFRt15-TGFRs were able to increase mitochondrial respiration in spleen cells. However, TGFRt15-TGFRs treated aged mice were able to increase the rate of basal respiration, ATP-related respiration, maximum oxygen consumption and reserve capacity to levels comparable to or higher than those observed in TGFRt15-TGFRs treated young mice spleen cells. These findings indicate that IL-15 immunostimulatory and TGF-beta antagonist activity of TGFRt15-TGFRs effectively stimulate and restore the immunocytopenic metabolic activity of aging mice.

⁺ Example 124: IL-15 Activity of TGFRt15-TGFRs in increasing CD 8T cells and NK cells

Six week old (young) and 72 week old (old) C57BL/6 mice were purchased from jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg) and TGFRt15-TGFRs (3 mg/kg) (n=6/group). On day 4 after treatment, mouse blood was collected from the submandibular vein in EDTA-containing tubes to assess the change in different immune cell subsets. Whole blood RBCs were lysed in ACK buffer for 5min at room temperature. The remaining cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To assess different types of immune cells in blood, cells were stained with CD3, CD4, CD45, CD8 and NK1.1 antibodies (hundred-in biotechnology) that specifically bound to the cell surface for 30min at Room Temperature (RT). After surface staining, cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5min at RT at 1500 RPM. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in FIG. 260, the results demonstrate that treatment of old mice with TGFRt15-TGFRs induced CD3 in the blood ⁺ CD45 ⁺ 、CD3 ⁺ CD8 ⁺ And CD3 ^- NK1.1 ⁺ The percentage of immune cells increased, whereas treatment of aged mice with TGFRt15 x-TGFRs had no effect on the percentage of these blood cell populations. These results indicate that IL-15 activity of TGFRt15-TGFRs increases CD8 in blood of aged mice ⁺ T cells and NK cells play a role. The old control mice had lower percentages of blood T cells and NK cells than the young control mice. However, treatment of CD3 in blood of aged mice with TGFRt15-TGFRs ⁺ CD45 ⁺ 、CD3 ⁺ CD8 ⁺ And CD3 ^- NK1.1 ⁺ The percentage of immune cells was increased to a level similar to that observed in blood treated with TGFRt15-TGFRs in young mice.

Six week old (young) and 72 week old (old) C57BL/6 mice were purchased from jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were treated subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg) and TGFRt15-TGFRs (3 mg/kg) (n=6/group). Four days after treatment, mice were euthanized, spleens were harvested and processed into single cell suspensions. Single cell suspensions were prepared to evaluate different sub-populations of immune cells. RBCs were lysed in ACK buffer for 5min at room temperature. The remaining cells were washed in FACS buffer (1X PBS (sea clone), containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)). To evaluate different types of immune cells in the spleen, cells were stained with CD3, CD45, CD8 and NK1.1 antibodies (hundred biotechnology) that specifically bound to the cell surface for 30 minutes at RT. After surface staining, cells were washed in FACS buffer (1X PBS (sea clone) containing 0.5% bsa (EMD milbo) and 0.001% sodium azide (sigma)) for 5min at 1500RPM at room temperature. After washing twice, the cells were resuspended in fixation buffer and analyzed by flow cytometry (Celesta-BD bioscience).

As shown in FIG. 261, the results demonstrate that treatment of aged mice with TGFRt15-TGFRs induced CD3 in the spleen ⁺ CD45 ⁺ 、CD3 ⁺ CD8 ⁺ And CD3 ^- NK1.1 ⁺ The percentage of immune cells increased, whereas treatment of aged mice with TGFRt15 x-TGFRs had no effect on the percentage of these spleen cell populations. These results indicate that IL-15 activity of TGFRt15-TGFRs increases CD8 in blood of aged mice ⁺ T cells and NK cells play a role. The spleen T cell and NK cell percentages were lower in the aged control mice than in the young control mice. However, treatment of aged mice with TGFRt15-TGFRs resulted in the incorporation of CD3 in the spleen ⁺ CD45 ⁺ 、CD3 ⁺ CD8 ⁺ And CD3 ^- NK1.1 ⁺ The percentage of immune cells increased to a level of young mice similar to that observed in spleens treated with TGFRt 15-TGFRs.

Example 125: TGFRt15-TGFRs are associated with naturally occurring aging cytopenia in the liver

Seventy-two week old (aged) C57BL/6 mice were purchased from jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were treated subcutaneously with PBS or one or two doses (on day 0 and day 60) of TGFRt15-TGFRs (3 mg/kg) (n=8/group). On day 71 post-treatment, will be smallMice were euthanized, and livers were harvested and stored in liquid nitrogen in 1.7mL Eppendorf tubes. Tissue samples were homogenized in 1mL Trizol (sammer femto) by using a homogenizer. The homogenized tissue was transferred to fresh Eppendorf tubes and total RNA was extracted using RNeasy Mini kit (kejie # 74106) according to the manufacturer's instructions. 1. Mu.g of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Kaiji). Real-time PCR was performed with a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers from Thermo Scientific. Reactions were performed in triplicate for all genes tested. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize the variability of gene expression levels. Relative to 18S rRNA, the expression of each target mRNA was calculated to be 2 based on Ct ^–Δ(ΔCt) Wherein Δct=ct _Target(s) –Ct _18S . Untreated 6 week old mice were used as controls to compare gene expression levels with aged mice. The results showed that the gene expression of IL-1α, IL-1β, IL-6, p21 and PAI-1 in the liver increased with age of mice, as expected, with age-dependent increase of cell senescence-associated transcripts. Treatment of mice 72 weeks old with single or two doses of TGFRt15-TGFRs resulted in a significant decrease in gene expression of the senescence markers IL-1α, IL-1β, IL-6, p21 and PAI-1 in the liver compared to PBS control (figure 262). These findings indicate that naturally occurring senile cytopenia in the liver of aged mice is associated with TGFRt 15-TGFRs.

Example 126: TGFRt15-TGFRs treatment reduces inflammation in liver tissue

Seventy-two week old (aged) C57BL/6 mice were purchased from jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were treated subcutaneously with PBS or one or two doses of TGFRt15-TGFRs (3 mg/kg) (n=10/group). On day 120 post-treatment, mice were sacrificed and mouse livers were prepared for histochemical evaluation. Liver tissue samples were fixed in 10% formaldehyde and after paraffin embedding procedure, cross sections were stained with hematoxylin-eosin. The extent of liver injury was assessed histologically in a blind manner. Histological sections of the whole liver region were scored for inflammation using a scale of 0 to 4 (0, absent and normal looking; 1, mild, 2, moderate, 3, severe, and 4, strong). As shown in figure 263, two doses of TGFRt15-TGFRs reduced the hepatitis score in the liver of aged mice compared to a single dose of TGFRt15-TGFRs or PBS control. These results indicate that TGFRt15-TGFRs treatment can reduce inflammation of liver tissue in aged mice.

Example 127: TGFRt15-TGFRs treatment decreases IL 1-alpha, IL-6, IL-8, PAI-1 and fibronectin levels

Seventy-two week old (aged) C57BL/6 mice were purchased from jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were treated with PBS or one or two doses (on day 0 and day 60) of TGFRt15-TGFRs (3 mg/kg) (n=10/group). On day 120 post-treatment, mice were euthanized, livers were harvested and stored in liquid nitrogen in 1.7mL Eppendorf tubes. Tissue samples were homogenized in 0.3mL of extraction buffer (Ai Bokang) by using a homogenizer. The homogenized tissue was transferred to fresh Eppendorf tubes. Protein levels in homogenized tissues were quantified using BCA protein assay kit (pierce). ELISA was performed using 25 μg of tissue homogenates to detect IL-1α, IL-1β, IL-6, IL-8, TGF- β, PAI-1, collagen and fibronectin (Andi organism). As shown in FIG. 264, the protein levels of IL-1α, IL-6, IL-8, PAI-1 and fibronectin were reduced in the livers of mice treated with 2 doses of TGFRt15-TGFRs compared to the PBS control group or the dose of TGFRt15-TGFRs treatment group. These results indicate that 2 doses of TGFRt15-TGFRs treatment can reduce the protein levels of IL-1α, IL-6, IL-8, PAI-1 and fibronectin in the liver of older mice. IL-1β, TGF- β and collagen protein levels were also lower in livers of mice treated with 2 doses of TGFRt15-TGFRs compared to PBS control; however, these changes did not reach statistical significance.

Example 128: TGFRt15-TGFRs reduce senescent cells

Seventy-two week old (aged) C57BL/6 mice were purchased from jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were treated subcutaneously with PBS or TGFRt15-TGFRs (3 mg/kg) (n=5/group). Day 4 after treatment, willMice were euthanized, livers were harvested, homogenized in PBS containing 2% fbs, and filtered in a 70 micron filter to obtain single cell suspensions. The cells were centrifuged and then resuspended in 5mL RPMI containing 0.5mg/mL collagenase IV and 0.02mg/mL DNAse in a 14mL round bottom tube. The cells were then shaken on a cyclotron shaker for 1 hour at 37℃and washed twice with RPMI. Cells were counted and at 2x10 ⁶ 2mL of complete medium (RPMI 1640 (Ji Bike), supplemented with 2mM L-glutamine (Semer Life technologies), penicillin (Semer Life technologies), streptomycin (Semer Life technologies) and 10% FBS (sea clone)) resuspended in 24 well flat bottom plate and incubated at 37℃in 5% CO ₂ Is cultured for 48 hours. Cells were harvested and washed once in warm complete medium at 1000rpm for 10 minutes at room temperature. The cell pellet was resuspended in 500. Mu.L of fresh medium, containing 1.5. Mu.L of senescence dye per tube (Ai Bokang). Cells were incubated at 37℃with 5% CO ₂ For 1-2 hours and washed twice with 500 μl wash buffer. The cell pellet was resuspended in 500. Mu.L of wash buffer and analyzed immediately by flow cytometry (Celesta-BD bioscience). As shown in FIG. 265, senescence marker β -gal 4 days after in vivo treatment with TGFRt15-TGFRs ⁺ The percentage of cells decreases. These results indicate that TGFRt15-TGFRs are able to reduce senescent cells (based on β -gal markers) in the liver of aged mice.

Example 129: effect of TGFRt15-TGFRs on survival of aged mice

Seventy-two week old C57BL/6 mice were purchased from Jackson laboratories. Mice were housed in a temperature and light controlled environment. Mice were treated subcutaneously with PBS or a dose of TGFRt15-TGFRs (3 mg/kg) (n=20/group). Mice survival was monitored daily until 120 weeks after treatment. The survival probability of the treatment group is shown in figure 266 based on the Mantel-Cox log rank test. Higher mortality was found in control mice compared to TGFRt15-TGFRs, as evidenced by a decrease in survival of the mice. By week 120 after treatment, the mortality rate of PBS control mice was 70%, while the mortality rate of TGFRt15-TGFRs treated mice was 45%.

Example 130: TGFRt15-TGFRs reduces SASP factor in liver of B16F10 tumor bearing mice following chemotherapy Is composed of (1) a base and (2) a plurality of base

The effect of TGFRt15-TGFRs treatment on reducing the level of SASP factor protein in B16F10 tumor bearing mice following chemotherapy was further evaluated. B16F10 tumor cells (1 x10 on day 0 ⁷ Individual cells/mice) were implanted into mice. Mice were treated subcutaneously with 10mg/kg docetaxel on days 1, 4 and 7. On day 8, mice were treated subcutaneously with PBS or TGFRt15-TGFRs (3 mg/kg). On day 17 post tumor inoculation, mice were euthanized, and livers were collected and homogenized. Protein levels of SASP factor in liver homogenates were determined by ELISA. As shown in figure 267, in vivo treatment with TGFRt15-TGFRs resulted in significant reduction of liver IL-1α, IL-6, tnfα and IL-8SASP factor levels following chemotherapy in B16F10 tumor bearing mice.

Example 131: TGFRt15-TGFRs mediated attenuation of immune cell subsets in B16F10 melanoma mouse model Function in eliminating old tumor cells

To assess the role of immune cell subsets in TGFRt15-TGFRs mediated elimination of senescent tumor cells, in vitro docetaxel-induced senescent B16F10-GFP tumor cells were mixed with parental B16F10 cells and subcutaneously implanted in mice after treatment with anti-NK 1.1 or anti-CD 8a antibodies. When the tumor reaches about 350mm ³ At this time, mice were randomly subjected to subcutaneous treatment with PBS or TGFRt15-TGFRs (3 mg/kg) +TA99 (200 μg). Mice were sacrificed on day 4 post-therapy and tumors were collected and analyzed. Assessment of GFP-positive B16F10-GFP TIS cells, NK and CD8 in tumors by flow cytometry ⁺ T cell level. As shown in figure 268A, the mixed tumors without immune depletion or cd8+ T immune cell depletion treated with TGFRt15-TGFRs contained significantly fewer senescent tumor cells expressing GFP than control treated mice. CD8 was also observed ⁺ Tumors of the depleted mice were significantly infiltrated with NK cells, and tumors of NK-depleted mice were significantly infiltrated with CD8 ⁺ T cell infiltration (figure 268B). These results indicate that NK and CD8 ⁺ T cells all play a role in controlling tumor growth, where NK cells are fineCells mediate mainly the activity of TGFRt15-TGFRs to deplete TIS tumor cells.

Example 132: anti-PD-L1 antibodies in combination with TGFRt15-TGFRs+TA99 in B16F10 melanoma mouse model And chemotherapy

To further evaluate the sequential TGFRt 15-TGFRs-immune checkpoint inhibitor treatment regimen (described in example 109), B16F10 tumor bearing mice were first treated with Docetaxel (DTX), then with anti-PD-L1 antibody followed by TGFRt15-tgfrs+ta99, or with TGFRt15-tgfrs+ta99 followed by anti-PD-L1 antibody (fig. 269A). Tumor growth curves and endpoint tumor volumes on day 18 indicated that both combination strategies (first with anti-PD-L1 antibody followed by TGFRt15-tgfrs+ta99 and trans-exchange before and after) showed a significant decrease in tumor volume compared to immunotherapy alone (TGFRt 15-tgfrs+ta99 or anti-PD-L1 alone) or DTX alone (fig. 269B). Interestingly, tumors treated with TGFRt15-TGFRs+TA99 showed significantly lower tumor volumes on day 13 before the start of the combination treatment compared to tumors treated with anti-PD-L1, indicating a role of TGFRt15-TGFRs+TA99 in the initial control of tumor growth. Endpoint analysis also showed that tumors treated with the combination of TGFRt15-tgfrs+ta99 and anti-PD-L1 antibody resulted in CD8 tumor infiltration compared to the single treatment group ⁺ T cell and NK cell levels are significantly increased. Combination therapy increased CD8 compared to monotherapy ⁺ Expression of the co-stimulatory receptor CD28 on TILs, suggesting that checkpoint blockade may rescue dysfunctional CD8 ⁺ TILs, which were further activated by IL-15 activity of TGFRt15-TGFRs in tumor microenvironment (fig. 269C). This was accompanied by spleen CD8 from the combination treatment group after stimulation with PMA/ionomycin ⁺ The enhanced activation phenotype (ifnγ secretion) of T cells (fig. 269D). The combination treatment also showed an increase in total CD8 in tumors compared to the immunotherapy treatment alone ⁺ T cells and CD44 ^hi CD8 ⁺ NKG2D expression by T cells (fig. 269E). Together, these data indicate that combined treatment of TGFRt15-tgfrs+ta99 and anti-PD-L1 antibody results in CD8 ⁺ Activation and infiltration of T cells, which may help to effectively control tumors.

Example 133: TGFRt15-TGFRs association against SW1990 human pancreatic tumor in C57BL/6SCID mice Antitumor efficacy of chemotherapy

To further evaluate the antitumor activity of TGFRt15-TGFRs combination chemotherapy, SW1990 human pancreatic cancer cells (2 x10 ⁶ Individual cells/mice) were injected subcutaneously (s.c.) into C57BL/6scid mice. Nine days after tumor cell implantation, chemotherapy with gemcitabine (40 mg/kg, i.p.) and albumin paclitaxel (5 mg/kg, i.p.) was started, followed by 2 days later by TGFRt15-TGFRs (3 mg/kg, s.c.). This is considered as one processing cycle, and is repeated for another 3 cycles (1 cycle/week) (fig. 270A). Tumor bearing control groups received PBS, chemotherapy or TGFRt15-TGFRs alone. Tumor volumes were measured and evaluated based on during and after study treatment <4000mm ³ Animal survival rate of tumor volume of (c). The results indicate that animals receiving the combination of TGFRt15-TGFRs and chemotherapy had significantly slower SW1990 tumor growth compared to PBS group (figures 270B-270C). TGFRt15-TGFRs+ chemotherapy also prolonged survival in SW1990 tumor-bearing mice (FIG. 270D). These results indicate that TGFRt15-TGFRs enhance the efficacy of standard care chemotherapy on human pancreatic tumors in a mouse xenograft tumor model.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Exemplary embodiments

Embodiment a1. A single chain chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A second target binding domain.

Embodiment a2. The single chain chimeric polypeptide of embodiment A1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other.

Embodiment a3. The single-chain chimeric polypeptide of embodiment A1, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain.

Embodiment a4. The single chain chimeric polypeptide of embodiments A1 to A3, wherein the soluble tissue factor domain and the second target binding domain are directly adjacent to each other.

Embodiment a5. The single-chain chimeric polypeptide of any one of embodiments A1 to A3, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain.

Embodiment a6. The single-chain chimeric polypeptide of embodiment A1, wherein the first target binding domain and the second target binding domain are directly adjacent to each other.

Embodiment A7. the single-stranded chimeric polypeptide of embodiment A1, wherein the single-stranded chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain.

Embodiment A8. the single stranded chimeric polypeptide of embodiment A6 or A7, wherein the second target binding domain and the soluble tissue factor domain are directly adjacent to each other.

Embodiment A9. the single-chain chimeric polypeptide of embodiment A6 or A7, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the second target binding domain and the soluble tissue factor domain.

Embodiment a10 the single chain chimeric polypeptide of any one of embodiments A1 to A9, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

Embodiment a11. The single chain chimeric polypeptide of embodiment a10, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment a12. The single chain chimeric polypeptide of embodiment a11, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment a13 the single chain chimeric polypeptide of any one of embodiments A1 to A9, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

Embodiment a14 the single chain chimeric polypeptide of any one of embodiments A1 to a13, wherein one or both of the first target binding domain and the second target binding domain is an antigen binding domain.

Embodiment a15 the single chain chimeric polypeptide of embodiment a14, wherein the first target binding domain and the second target binding domain are each an antigen binding domain.

Embodiment a16. The single chain chimeric polypeptide of embodiment a13, wherein the antigen binding domain comprises an scFv or a single domain antibody.

Embodiment a17 the single chain chimeric polypeptide of any one of embodiments A1 to a16, wherein one or both of the first target binding domain and the second target binding domain bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC A, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-. Beta.receptor II (TGF-. Beta.RII), ligands of DNAM1, ligands of NKp46, ligands of NKp44 a ligand for NKG2D, a ligand for NKp30, a ligand for scmhc i, a ligand for scmhc II, a ligand for scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-D, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD 28.

Embodiment a18 the single chain chimeric polypeptide of any one of embodiments A1 to a16, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein.

Embodiment a19 the single chain chimeric polypeptide of embodiment a18, wherein the soluble interleukin, cytokine or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-D and SCF, FLT3L, MICA, MICB, and ULP16 binding protein.

Embodiment a20 the single chain chimeric polypeptide of any one of embodiments A1 to a16, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor.

Embodiment a21 the single chain chimeric polypeptide of embodiment a20, wherein the soluble interleukin or cytokine receptor is soluble TGF-beta receptor II (TGF- βrii), soluble TGF- βriii, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28.

Embodiment a22 the single chain chimeric polypeptide of any one of embodiments A1 to a21, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment A23 the single chain chimeric polypeptide of embodiment A22, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment a24. The single chain chimeric polypeptide of embodiment a23, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID No. 93.

Embodiment A25 the single chain chimeric polypeptide of embodiment A24, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment a26. The single chain chimeric polypeptide of any one of embodiments a22 to a25, wherein the soluble human tissue factor domain does not comprise one or more of:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

Arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment a27. The single chain chimeric polypeptide of embodiment a26, wherein the soluble human tissue factor domain does not comprise any of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment a28 the single chain chimeric polypeptide of any one of embodiments A1 to a27, wherein the soluble tissue factor domain is incapable of binding factor vila.

Embodiment a29 the single chain chimeric polypeptide of any one of embodiments A1 to a28, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment a30 the single chain chimeric polypeptide of any one of embodiments A1 to a29, wherein the single chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment a31 the single-chain chimeric polypeptide of any one of embodiments A1 to a30, wherein the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and/or C-terminus.

Embodiment a32. The single-chain chimeric polypeptide of embodiment a31, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus.

Embodiment a33. The single-chain chimeric polypeptide of embodiment a32, wherein one or more additional target binding domains directly adjoin the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment a34. The single-chain chimeric polypeptide of embodiment a33, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domains and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment a35 the single-chain chimeric polypeptide of embodiment a31, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its C-terminus.

Embodiment a36. The single-chain chimeric polypeptide of embodiment a35, wherein one of the one or more additional target binding domains directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment a37. The single-chain chimeric polypeptide of embodiment a35, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domains and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment a38. The single-chain chimeric polypeptide of embodiment a31, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus and C-terminus.

Embodiment a39. The single chain chimeric polypeptide of embodiment a38, wherein one of the one or more additional antigen binding domains at the N-terminus directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment a40. The single-chain chimeric polypeptide of embodiment a38, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains and the first target binding domain, the second target binding domain, or the soluble tissue factor domain at the N-terminus.

Embodiment a41. The single chain chimeric polypeptide of embodiment a38, wherein one of the one or more additional antigen binding domains at the C-terminus directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment a42. The single-chain chimeric polypeptide of embodiment a38, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains and the first target binding domain, the second target binding domain, or the soluble tissue factor domain at the C-terminus.

Embodiment a43 the single chain chimeric polypeptide of any one of embodiments a31 to a42, wherein two or more of the first target binding domain, the second target binding domain and one or more additional target binding domains specifically bind to the same antigen.

Embodiment a44. The single-chain chimeric polypeptide of embodiment a43, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope.

Embodiment a45 the single chain chimeric polypeptide of embodiment a44, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

Embodiment a46. The single-chain chimeric polypeptide of embodiment a43, wherein the first target binding domain, the second target binding domain, and one or more additional target binding domains each specifically bind to the same antigen.

Embodiment a47. The single-chain chimeric polypeptide of embodiment a46, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope.

Embodiment a48 the single chain chimeric polypeptide of embodiment a47, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

Embodiment a49 the single chain chimeric polypeptide of any one of embodiments a31 to a42, wherein the first target binding domain, the second target binding domain and one or more additional target binding domains specifically bind to different antigens.

Embodiment a50. The single chain chimeric polypeptide of any one of embodiments a31 to a49, wherein one or more of the first target binding domain, the second target binding domain and the one or more target binding domains are antigen binding domains.

Embodiment a51 the single chain chimeric polypeptide of embodiment a50, wherein the first target binding domain, the second target binding domain and the one or more additional target binding domains are each antigen binding domains.

Embodiment a52 the single chain chimeric polypeptide of embodiment a51, wherein the antigen binding domain comprises an scFv or a single domain antibody.

Embodiment a53. The single-chain chimeric polypeptide of any one of embodiments a31 to a52, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC A, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-. Beta.receptor II (TGF-. Beta.RII), ligands of DNAM1, ligands of NKp46, ligands of NKp44 a ligand for NKG2D, a ligand for NKp30, a ligand for scmhc i, a ligand for scmhc II, a ligand for scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-D, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD 28.

Embodiment a54. The single chain chimeric polypeptide of any one of embodiments a31 to a52, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment a55. The single chain chimeric polypeptide of embodiment a54, wherein the soluble interleukin, cytokine or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-D and SCF, FLT3L, MICA, MICB, and ULP16 binding protein.

Embodiment a56. The single chain chimeric polypeptide of any one of embodiments a31 to a52, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment a57 the single chain chimeric polypeptide of embodiment a56, wherein the soluble receptor is soluble TGF- β receptor II (TGF- βrii), soluble TGF- βriii, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.

Embodiment a58 the single-chain chimeric polypeptide of any one of embodiments A1 to a57, wherein the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment a59 the single-chain chimeric polypeptide of any one of embodiments A1 to a58, wherein the single-chain chimeric polypeptide further comprises a peptide tag at the N-terminus or the C-terminus of the single-chain chimeric polypeptide.

Embodiment a60. A composition comprising any one of the single-chain chimeric polypeptides of embodiments A1 to a59.

Embodiment a61 the composition of embodiment a60, wherein the composition is a pharmaceutical composition.

Embodiment a62. A kit comprising at least one dose of the composition of embodiment a60 or a61.

Embodiment a63. A nucleic acid encoding any one of the single-chain chimeric polypeptides of any one of embodiments A1 to a59.

Embodiment a64 a vector comprising the nucleic acid of embodiment a63.

Embodiment a65 the vector of embodiment a64, wherein the vector is an expression vector.

Embodiment a66. A cell comprising the nucleic acid of embodiment a63 or the vector of embodiment a64 or a65.

Embodiment a67. A method of producing a single-chain chimeric polypeptide, the method comprising:

culturing the cells of embodiment a66 in a culture medium under conditions sufficient to cause production of the single-chain chimeric polypeptide; and

Recovering the single-chain chimeric polypeptide from the cells and/or the culture medium.

Embodiment a68 a single chain chimeric polypeptide produced by the method of embodiment a 67.

Embodiment a69. The single chain chimeric polypeptide of embodiment a26, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 97.

Embodiment A70. The single chain chimeric polypeptide of embodiment A69, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 97.

Embodiment A71 the single chain chimeric polypeptide of embodiment A70, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 97.

Embodiment A72 the single chain chimeric polypeptide of embodiment B71, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO 97.

Embodiment a73. The single chain chimeric polypeptide of embodiment a26, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 98.

Embodiment a74 the single chain chimeric polypeptide of embodiment a73, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID No. 98.

Embodiment A75. The single chain chimeric polypeptide of embodiment A74, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 98.

Embodiment A76. The single chain chimeric polypeptide of embodiment B60, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO 98.

Embodiment b1. A single chain chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A second target binding domain that is complementary to the first target binding domain,

wherein:

the first target binding domain and the second target binding domain each specifically bind to an IL-2 receptor; or (b)

The first target binding domain and the second target binding domain each specifically bind to an IL-15 receptor.

Embodiment B2. The single chain chimeric polypeptide of embodiment B1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other.

Embodiment B3. The single-chain chimeric polypeptide of embodiment B1, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain.

Embodiment B4. the single stranded chimeric polypeptide of embodiments B1 to B3 wherein the soluble tissue factor domain and the second target binding domain are directly adjacent to each other.

Embodiment B5. the single-chain chimeric polypeptide of any one of embodiments B1 to B3, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain.

Embodiment B6. the single stranded chimeric polypeptide of embodiment B1 wherein the first target binding domain and the second target binding domain are directly adjacent to each other.

Embodiment B7. the single-stranded chimeric polypeptide of embodiment B1, wherein the single-stranded chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain.

Embodiment B8. the single stranded chimeric polypeptide of embodiment B6 or B7, wherein the second target binding domain and the soluble tissue factor domain are directly adjacent to each other.

Embodiment B9. the single-chain chimeric polypeptide of embodiment B6 or B7, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the second target binding domain and the soluble tissue factor domain.

Embodiment B10. The single chain chimeric polypeptide of any one of embodiments B1 to B9, wherein both the first target binding domain and the second target binding domain are soluble interleukin proteins.

Embodiment B11. The single chain chimeric polypeptide of embodiment B10, wherein the first target binding domain and the second target binding domain are soluble IL-2 proteins.

Embodiment B12. The single chain chimeric polypeptide of embodiment B11, wherein the soluble IL-2 protein is a soluble human IL-2 protein.

Embodiment B13. The single chain chimeric polypeptide of embodiment B12, wherein the soluble human IL-2 protein comprises SEQ ID NO:78.

Embodiment B14. The single chain chimeric polypeptide of embodiment B10, wherein the first target binding domain and the second target binding domain are soluble IL-15 proteins.

Embodiment B15 the single chain chimeric polypeptide of embodiment B14, wherein the soluble IL-15 protein is a soluble human IL-15 protein.

Embodiment B16. The single chain chimeric polypeptide of embodiment B15, wherein the soluble human IL-15 protein comprises SEQ ID NO. 82.

Embodiment B17 the single chain chimeric polypeptide of any one of embodiments B1 to B16, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment B18. The single chain chimeric polypeptide of embodiment B17, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment B19. The single chain chimeric polypeptide of embodiment B18, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 93.

Embodiment B20. The single chain chimeric polypeptide of embodiment B19, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment B21. The single chain chimeric polypeptide of any one of embodiments B17 to B20, wherein the soluble human tissue factor domain does not comprise one or more of:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment B22. The single chain chimeric polypeptide of embodiment B21, wherein the soluble human tissue factor domain does not comprise any of the following:

Lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment B23 the single chain chimeric polypeptide of any one of embodiments B1 to B22, wherein the soluble tissue factor domain is incapable of binding factor vila.

Embodiment B24 the single chain chimeric polypeptide of any one of embodiments B1 to B23, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment B25 the single-chain chimeric polypeptide of any one of embodiments B1 to B24, wherein the single-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment B26. The single-chain chimeric polypeptide of any one of embodiments B1 to B25, wherein the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and/or C-terminus.

Embodiment B27. The single-chain chimeric polypeptide of embodiment B26, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus.

Embodiment B28. The single-chain chimeric polypeptide of embodiment B27, wherein one or more additional target binding domains directly adjoin the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment B29. The single-chain chimeric polypeptide of embodiment B28, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment B30. The single-chain chimeric polypeptide of embodiment B26, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its C-terminus.

Embodiment B31. The single-chain chimeric polypeptide of embodiment B30, wherein one of the one or more additional target binding domains directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment B32. The single-chain chimeric polypeptide of embodiment B30, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment B33. The single-chain chimeric polypeptide of embodiment B26, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-and C-terminus.

Embodiment B34. The single chain chimeric polypeptide of embodiment B33, wherein one of the one or more additional antigen binding domains at the N-terminus directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment B35 the single chain chimeric polypeptide of embodiment B33, wherein the single chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains and the first target binding domain, the second target binding domain or the soluble tissue factor domain at the N-terminus.

Embodiment B36. The single chain chimeric polypeptide of embodiment B33, wherein one of the one or more additional antigen binding domains at the C-terminus directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment B37. The single-chain chimeric polypeptide of embodiment B33, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains and the first target binding domain, the second target binding domain, or the soluble tissue factor domain at the C-terminus.

Embodiment B38. The single chain chimeric polypeptide of any one of embodiments B26 to B37, wherein each of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically binds to an IL-2 receptor or an IL-15 receptor.

Embodiment B39. The single-chain chimeric polypeptide of embodiment B38, wherein each of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprises the same amino acid sequence.

Embodiment B40. The single chain chimeric polypeptide of any one of embodiments B26 to B37, wherein one or more additional target binding domains are antigen binding domains.

Embodiment B41. The single chain chimeric polypeptide of embodiment B40, wherein the antigen binding domain comprises an scFv or a single domain antibody.

Embodiment B42. The single chain chimeric polypeptide of any one of embodiments B26 to B37, B40 and B41, wherein the one or more additional target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-beta receptor II (TGF-. Beta.RII), ligands of DNAM-1, ligands of TGF-46, TGF-beta-RIII ligands for NKp44, NKG2D, NKp30, scMHCI, scMHCII, scTCR, IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, stem Cell Factor (SCF), stem cell-like tyrosine kinase 3 (FLT 3L), MICA, MICB, ULP16 binding protein, CD155, CD122 and CD 28.

Embodiment B43 the single chain chimeric polypeptide of any one of embodiments B6 to B37, B40 and B41, wherein the one or more additional target binding domains are soluble interleukins or cytokine proteins.

Embodiment B44. The single chain chimeric polypeptide of embodiment B43, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

Embodiment B45 the single chain chimeric polypeptide of any one of embodiments B6 to B37, B40 and B41, wherein one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment B46. The single chain chimeric polypeptide of embodiment B45, wherein the soluble receptor is soluble TGF- β receptor II (TGF- βrii) and soluble TGF- βriii.

Embodiment B47. The single-chain chimeric polypeptide of any one of embodiments B1 to B46, wherein the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment B48 the single-chain chimeric polypeptide of any one of embodiments B1 to B47, wherein the single-chain chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the single-chain chimeric polypeptide.

Embodiment B49 a composition comprising any one of the single-chain chimeric polypeptides of embodiments B1 to B48.

Embodiment B50 the composition of embodiment B49, wherein the composition is a pharmaceutical composition.

Embodiment B51 a kit comprising at least one dose of the composition of embodiment B49 or B50.

Embodiment B52. A nucleic acid encoding any one of the single-chain chimeric polypeptides of any one of embodiments B1 to B48.

Embodiment B53 a vector comprising the nucleic acid of embodiment B52.

Embodiment B54 the vector of embodiment B53, wherein the vector is an expression vector.

Embodiment B55 a cell comprising the nucleic acid of embodiment B52 or the vector of embodiment B53 or B54.

Embodiment b56. A method of producing a single-chain chimeric polypeptide, the method comprising:

culturing the cells of embodiment B55 in a culture medium under conditions sufficient to cause production of the single-chain chimeric polypeptide; and

recovering the single-chain chimeric polypeptide from the cells and/or the culture medium.

Embodiment B57 a single chain chimeric polypeptide produced by the method of embodiment B56.

Embodiment B58 the single chain chimeric polypeptide of embodiment B21, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 97.

Embodiment B59 the single chain chimeric polypeptide of embodiment B58, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 97.

Embodiment B60. Single-chain chimeric polypeptide of embodiment B59, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 97.

Embodiment B61 the single chain chimeric polypeptide of embodiment B60, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO 97.

Embodiment B62. The single chain chimeric polypeptide of embodiment B21, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 98.

Embodiment B63. The single chain chimeric polypeptide of embodiment B62, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 98.

Embodiment B64. The single chain chimeric polypeptide of embodiment B63, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 98.

Embodiment B65. The single chain chimeric polypeptide of embodiment B64, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO. 98.

Embodiment c1. A multi-chain chimeric polypeptide comprising:

(a) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(b) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of a first domain and a second domain of a pair of affinity domains.

Embodiment C2. the multi-chain chimeric polypeptide of embodiment C1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

Embodiment C3. the multi-chain chimeric polypeptide of embodiment C1, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment C4. The multi-chain chimeric polypeptide of any one of embodiments C1 to C3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

Embodiment C5. the multi-chain chimeric polypeptide of any one of embodiments C1 to C3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment C6. the multi-chain chimeric polypeptide of any one of embodiments C1 to C5, wherein the second domain of a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

Embodiment C7. the multi-chain chimeric polypeptide of any one of embodiments C1 to C5, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

Embodiment C8. the multi-chain chimeric polypeptide of any one of embodiments C1 to C7, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

Embodiment C9. the multi-chain chimeric polypeptide of embodiment C8, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment C10. The multi-chain chimeric polypeptide of embodiment C9, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment C11. The multi-chain chimeric polypeptide of any one of embodiments C1 to C7, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

Embodiment C12 the multi-chain chimeric polypeptide of any one of embodiments C1 to C11, wherein one or both of the first target binding domain and the second target binding domain is an antigen binding domain.

Embodiment C13. The multi-chain chimeric polypeptide of embodiment C12, wherein the first target binding domain and the second target binding domain are each an antigen binding domain.

Embodiment C14. The multi-chain chimeric polypeptide of embodiment C12 or C13, wherein the antigen binding domain comprises an scFv or single domain antibody.

Embodiment C15 the multi-chain chimeric polypeptide of any one of embodiments C1 to C14, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-beta receptor II (TGF-. Beta.RII), ligands of DNAM-1, ligands of TGF-46, TGF-beta-RIII ligands for NKp44, NKG2D, NKp30, scMHCI, scMHCII, scTCR, IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, stem Cell Factor (SCF), stem cell-like tyrosine kinase 3 (FLT 3L), MICA, MICB, ULP16 binding protein, CD155, CD122 and CD 28.

Embodiment C16 the multi-chain chimeric polypeptide of any one of embodiments C1 to C14, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein.

Embodiment C17. The multi-chain chimeric polypeptide of embodiment C16, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding proteins.

Embodiment C18 the multi-chain chimeric polypeptide of any one of embodiments C1 to C14, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor.

Embodiment C19 the multi-chain chimeric polypeptide of embodiment C18, wherein the soluble receptor is soluble TGF- β receptor II (TGF- βrii), soluble TGF- βriii, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28.

Embodiment C20. The multi-chain chimeric polypeptide of any one of embodiments C1 to C19, wherein the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more additional antigen binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment C21. The multi-chain chimeric polypeptide of embodiment C20, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more other antigen binding domains, and/or a linker sequence between at least one of the one or more other antigen binding domains and the first domain in the pair of affinity domains.

Embodiment C22. The multi-chain chimeric polypeptide of any one of embodiments C1 to C19, wherein the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and/or C-terminus of the first chimeric polypeptide.

Embodiment C23. The multi-chain chimeric polypeptide of embodiment C22, wherein in the first chimeric polypeptide at least one of the one or more additional target binding domains directly abuts the first domain in the pair of affinity domains.

Embodiment C24. The multi-chain chimeric polypeptide of embodiment C22, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains.

Embodiment C25. The multi-chain chimeric polypeptide of embodiment C22, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains directly adjoins the first target binding domain.

Embodiment C26. The multi-chain chimeric polypeptide of embodiment C22, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain.

Embodiment C27. The multi-chain chimeric polypeptide of embodiment C22, wherein at least one of the one or more additional target binding domains is disposed N-terminal and/or C-terminal to the first chimeric polypeptide, and at least one of the one or more additional target binding domains is located between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment C28. The multi-chain chimeric polypeptide of embodiment C27, wherein at least one of the one or more additional target binding domains disposed N-terminally in the first chimeric polypeptide directly abuts the first target binding domain or the first domain in a pair of affinity domains.

Embodiment C29. The multi-chain chimeric polypeptide of embodiment C27, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment C30. The multi-chain chimeric polypeptide of embodiment C27, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed at the C-terminus directly abuts the first target binding domain or the first domain of the pair of affinity domains.

Embodiment C31. The multi-chain chimeric polypeptide of embodiment C27, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment C32. The multi-chain chimeric polypeptide of embodiment C27, wherein at least one of the one or more additional target binding domains located between the soluble tissue factor domain and the first domain of the pair of affinity domains directly adjoins the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment C33. The multi-chain chimeric polypeptide of embodiment C27, wherein the first chimeric polypeptide further comprises a linker sequence disposed as follows: (i) Between the soluble tissue factor domain and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains; and/or (ii) between a first domain of the pair of affinity domains and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains being located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment C34 the multi-chain chimeric polypeptide of any one of embodiments C1 to C33, wherein the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment C35. The multi-chain chimeric polypeptide of embodiment C34, wherein in the second chimeric polypeptide at least one of the one or more additional target binding domains directly adjoins the second domain in the pair of affinity domains.

Embodiment C36. The multi-chain chimeric polypeptide of embodiment C34, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain in the pair of affinity domains.

Embodiment C37. The multi-chain chimeric polypeptide of embodiment C34, wherein in the second chimeric polypeptide, at least one of the one or more additional target binding domains directly adjoins the second target binding domain.

Embodiment C38. The multi-chain chimeric polypeptide of embodiment C34, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

Embodiment C39 the multi-chain chimeric polypeptide of any one of embodiments C20 to C38, wherein two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to the same antigen.

Embodiment C40. The multi-chain chimeric polypeptide of embodiment C39, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope.

Embodiment C41. The multi-chain chimeric polypeptide of embodiment C40, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

Embodiment C42. The multi-chain chimeric polypeptide of embodiment C39, wherein the first target binding domain, the second target binding domain, and one or more additional target binding domains each specifically bind to the same antigen.

Embodiment C43 the multi-chain chimeric polypeptide of embodiment C42, wherein the first target binding domain, the second target binding domain and the one or more additional target binding domains each specifically bind to the same epitope.

Embodiment C44. The multi-chain chimeric polypeptide of embodiment C43, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

Embodiment C45 the multi-chain chimeric polypeptide of any one of embodiments C20 to C38, wherein the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to different antigens.

Embodiment C46. The multi-chain chimeric polypeptide of any one of embodiments C20 to C45, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains.

Embodiment C47. The multi-chain chimeric polypeptide of embodiment C46, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains.

Embodiment C48 the multi-chain chimeric polypeptide of embodiment C47, wherein the antigen binding domain comprises an scFv.

Embodiment C49. The multi-chain chimeric polypeptide of any one of embodiments C20 to C48, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-beta receptor II (TGF-. Beta.RII), ligands of DNAM-1, ligands of NKp46, ligands of NKp44, TGF-. Beta.RIII ligands for NKG2D, ligands for NKG 30, ligands for scMHCI, ligands for scMHCII, ligands for scTCR, receptors for IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, stem Cell Factor (SCF), stem cell-like tyrosine kinase 3 ligand (FLT 3L), MICA, MICB, ULP16 binding protein, CD155, CD122 and CD3, and CD 28.

Embodiment C50. The multi-chain chimeric polypeptide of any one of embodiments C20 to C48, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment C51. The multi-chain chimeric polypeptide of embodiment C50, wherein the soluble interleukin, cytokine or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF, FLT3L, MICA, MICB, and ULP16 binding protein.

Embodiment C52 the multi-chain chimeric polypeptide of any one of embodiments C20 to C48, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment C53 the multi-chain chimeric polypeptide of embodiment C52, wherein the soluble receptor is soluble TGF-beta receptor II (TGF- βrii), soluble TGF- βriii, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.

Embodiment C54 the multi-chain chimeric polypeptide of any one of embodiments C1 to C53, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide.

Embodiment C55 the multi-chain chimeric polypeptide of any one of embodiments C1 to C53, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment C56 the multi-chain chimeric polypeptide of any one of embodiments C1 to C55, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment C57. The multi-chain chimeric polypeptide of embodiment C56, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93.

Embodiment C58 the multi-chain chimeric polypeptide of embodiment C57, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID No. 93.

Embodiment C59 the multi-chain chimeric polypeptide of embodiment C58, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment C60. The multi-chain chimeric polypeptide of any one of embodiments C56 to C59, wherein the soluble human tissue factor domain does not comprise one or more of:

Lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment C61. The multi-chain chimeric polypeptide of embodiment C60, wherein the soluble human tissue factor domain does not comprise any of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

Tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment C62 the multi-chain chimeric polypeptide of any one of embodiments C1 to C61, wherein the soluble tissue factor domain is incapable of binding to factor vila.

Embodiment C63 the multi-chain chimeric polypeptide of any one of embodiments C1 to C62, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment C64 the multi-chain chimeric polypeptide of any one of embodiments C1 to C63, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment C65 the multi-chain chimeric polypeptide of any one of embodiments C1 to C64, wherein the pair of affinity domains is a sushi domain from the human IL-15 receptor alpha chain (IL 15rα) and soluble IL-15.

Embodiment C66 the multi-chain chimeric polypeptide of embodiment C65, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment C67 the multi-chain chimeric polypeptide of embodiment C65 or C66, wherein the human IL-15Rα is mature full-length IL-15Rα.

Embodiment C68. The multi-chain chimeric polypeptide of any one of embodiments C1 to C64, wherein a pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

Embodiment C69 the multi-chain chimeric polypeptide of any one of embodiments C1 to C68, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment C70. A composition comprising any of the multiple chain chimeric polypeptides of embodiments C1 to C69.

Embodiment C71 the composition of embodiment C70, wherein the composition is a pharmaceutical composition.

Embodiment C72 a kit comprising at least one dose of the composition of embodiment C70 or C71.

Embodiment C73. A nucleic acid encoding any one of the multi-chain chimeric polypeptides of any one of embodiments C1 to C69.

Embodiment C74 a vector comprising the nucleic acid of embodiment C73.

Embodiment C75 the vector of embodiment C74, wherein the vector is an expression vector.

Embodiment C76 a cell comprising the nucleic acid of embodiment C73 or the vector of embodiment C74 or C75.

Embodiment c77. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cells of embodiment C76 in a culture medium under conditions sufficient to cause production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cells and/or culture medium.

Embodiment C78 a multi-chain chimeric polypeptide produced by the method of embodiment C77.

Embodiment C79 the multi-chain chimeric polypeptide of embodiment A56, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO 97.

Embodiment C80 the multi-chain chimeric polypeptide of embodiment C79, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO 97.

Embodiment C81 the multi-chain chimeric polypeptide of embodiment C80, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 97.

Embodiment C82 the multi-chain chimeric polypeptide of embodiment C81, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO 97.

Embodiment C83. The multi-chain chimeric polypeptide of embodiment C56, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 98.

Embodiment C84 the multi-chain chimeric polypeptide of embodiment C83, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 98.

Embodiment C85 the multi-chain chimeric polypeptide of embodiment C84, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID No. 98.

Embodiment C86 the multi-chain chimeric polypeptide of embodiment C85, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO. 98.

Embodiment d1. A multi-chain chimeric polypeptide comprising:

(a) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(b) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of a first domain and a second domain of a pair of affinity domains;

the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-18 or IL-12.

Embodiment D2. the multi-chain chimeric polypeptide of embodiment D1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

Embodiment D3. the multi-chain chimeric polypeptide of embodiment D1, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment D4. the multi-chain chimeric polypeptide of any one of embodiments D1 to D3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

Embodiment D5. the multi-chain chimeric polypeptide of any one of embodiments D1 to D3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment D6. the multi-chain chimeric polypeptide of any one of embodiments D1 to D5, wherein the second domain of a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

Embodiment D7. the multi-chain chimeric polypeptide of any one of embodiments D1 to D5, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

Embodiment D8. the multi-chain chimeric polypeptide of any one of embodiments D1 to D7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment D9. the multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment D10 the multi-chain chimeric polypeptide of embodiment D9, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 93.

Embodiment D11 the multi-chain chimeric polypeptide of embodiment D10, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment D12. The multi-chain chimeric polypeptide of any one of embodiments D8 to D11, wherein the soluble human tissue factor domain does not comprise one or more of the following:

Lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment D13. The multi-chain chimeric polypeptide of embodiment D12, wherein the soluble human tissue factor domain does not comprise any of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

Tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment D14 the multi-chain chimeric polypeptide of any one of embodiments D1 to D13, wherein the soluble tissue factor domain is not capable of binding to factor vila.

Embodiment D15 the multi-chain chimeric polypeptide of any one of embodiments D1 to D14, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment D16 the multi-chain chimeric polypeptide of any one of embodiments D1 to D15, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment D17 the multi-chain chimeric polypeptide of any one of embodiments D1 to D16, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide.

Embodiment D18 the multi-chain chimeric polypeptide of any one of embodiments D1 to D17, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment D19 the multi-chain chimeric polypeptide of any one of embodiments D1 to D18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment D20 the multi-chain chimeric polypeptide of embodiment D19, wherein the signal sequence comprises SEQ ID NO. 117.

Embodiment D21 the multi-chain chimeric polypeptide of embodiment D20, wherein the signal sequence is SEQ ID NO. 117.

Embodiment D22 the multi-chain chimeric polypeptide of any one of embodiments D1 to D21, wherein the pair of affinity domains is a sushi domain from the human IL-15 receptor alpha chain (IL-15 ra) and soluble IL-15.

Embodiment D23 the multi-chain chimeric polypeptide of embodiment D22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment D24 the multi-chain chimeric polypeptide of embodiment D22, wherein the soluble IL-15 comprises a sequence at least 80% identical to SEQ ID NO. 82.

Embodiment D25 the multi-chain chimeric polypeptide of embodiment D24, wherein the soluble IL-15 comprises a sequence at least 90% identical to SEQ ID NO. 82.

Embodiment D26 the multi-chain chimeric polypeptide of embodiment D25, wherein the soluble IL-15 comprises a sequence at least 95% identical to SEQ ID NO. 82.

Embodiment D27 the multi-chain chimeric polypeptide of embodiment D26, wherein the soluble IL-15 comprises SEQ ID NO. 82.

Embodiment D28 the multi-chain chimeric polypeptide of any one of embodiments D22 to D27, wherein the sushi domain of IL-15 ra comprises a sushi domain from human IL-15 ra.

Embodiment D29 the multi-chain chimeric polypeptide of embodiment D28, wherein the sushi domain from human IL-15Rα comprises a sequence 80% identical to SEQ ID NO 113.

Embodiment D30 the multi-chain chimeric polypeptide of embodiment D29, wherein the sushi domain from human IL-15Rα comprises a sequence 90% identical to SEQ ID NO 113.

Embodiment D31 the multi-chain chimeric polypeptide of embodiment D30, wherein the sushi domain from human IL-15Rα comprises a sequence 95% identical to SEQ ID NO 113.

Embodiment D32 the multi-chain chimeric polypeptide of embodiment D31, wherein the sushi domain from human IL-15Rα comprises SEQ ID NO 113.

Embodiment D33 the multi-chain chimeric polypeptide of embodiment D28, wherein the sushi domain from human IL-15Rα is mature full-length IL-15Rα.

Embodiment D34 the multi-chain chimeric polypeptide of any one of embodiments D1 to D21, wherein a pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

Embodiment D35 the multi-chain chimeric polypeptide of any one of embodiments D1 to D34, wherein one or both of the first target binding domain and the second target binding domain is an agonistic antigen binding domain.

Embodiment D36 the multi-chain chimeric polypeptide of embodiment D35, wherein the first target binding domain and the second target binding domain are each an agonistic antigen binding domain.

Embodiment D37 the multi-chain chimeric polypeptide of embodiment D35 or D36, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment D38 the multi-chain chimeric polypeptide of any one of embodiments D1 to D34, wherein one or both of the first target binding domain and the second target binding domain is soluble IL-15 or soluble IL-18.

Embodiment D39 the multi-chain chimeric polypeptide of embodiment D38, wherein the first target binding domain and the second target binding domain are each independently soluble IL-15 or soluble IL-18.

Embodiment D40 the multi-chain chimeric polypeptide of any one of embodiments D1 to D39, wherein both the first target binding domain and the second target binding domain specifically bind to a receptor for IL-18 or IL-12.

Embodiment d41 the multi-chain chimeric polypeptide of embodiment B40, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment D42 the multi-chain chimeric polypeptide of embodiment D41, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment D43 the multi-chain chimeric polypeptide of any one of embodiments D1 to D39, wherein the first target binding domain specifically binds to a receptor for IL-12 and the second target binding domain specifically binds to a receptor for IL-18.

Embodiment D44 the multi-chain chimeric polypeptide of any one of embodiments D1 to D39, wherein the first target binding domain specifically binds to a receptor for IL-18 and the second target binding domain specifically binds to a receptor for IL-12.

Embodiment D45 the multi-chain chimeric polypeptide of embodiment D44, wherein the first target binding domain comprises soluble IL-18.

Embodiment D46 the multi-chain chimeric polypeptide of embodiment D45, wherein the soluble IL-18 is soluble human IL-18.

Embodiment D47. The multi-chain chimeric polypeptide of embodiment D46, wherein the soluble human IL-18 comprises a sequence at least 80% identical to SEQ ID NO 109.

Embodiment D48 the multi-chain chimeric polypeptide of embodiment D47, wherein the soluble human IL-18 comprises a sequence at least 90% identical to SEQ ID NO 109.

Embodiment D49 the multi-chain chimeric polypeptide of embodiment D48, wherein the soluble human IL-18 comprises a sequence at least 95% identical to SEQ ID NO 109.

Embodiment D50 the multi-chain chimeric polypeptide of embodiment D49, wherein the soluble human IL-18 comprises the sequence of SEQ ID NO. 109.

Embodiment D51 the multi-chain chimeric polypeptide of any one of embodiments D44 to D50, wherein the second target binding domain comprises soluble IL-12.

Embodiment D52 the multi-chain chimeric polypeptide of embodiment D51, wherein the soluble IL-18 is soluble human IL-12.

Embodiment D53 the multi-chain chimeric polypeptide of embodiment D52, wherein the soluble human IL-15 comprises a sequence of soluble human IL-12β (p 40) and a sequence of soluble human IL-12α (p 35).

Embodiment D54 the multi-chain chimeric polypeptide of embodiment D53, wherein the soluble human IL-15 further comprises a linker sequence between the sequence of soluble IL-12β (p 40) and the sequence of soluble human IL-12α (p 35).

Embodiment D55 the multi-chain chimeric polypeptide of embodiment D54, wherein the linker sequence comprises SEQ ID NO. 102.

Embodiment D56 the multi-chain chimeric polypeptide of any one of embodiments D53 to D55, wherein the sequence of soluble human IL-12β (p 40) comprises a sequence at least 80% identical to SEQ ID NO. 81.

Embodiment D57 the multi-chain chimeric polypeptide of embodiment D56, wherein the sequence of soluble human IL-12β (p 40) comprises a sequence at least 90% identical to SEQ ID NO. 81.

Embodiment D58 the multi-chain chimeric polypeptide of embodiment D57, wherein the sequence of soluble human IL-12β (p 40) comprises a sequence at least 95% identical to SEQ ID NO. 81.

Embodiment D59 the multi-chain chimeric polypeptide of embodiment D58, wherein the sequence of soluble human IL-12β (p 40) comprises SEQ ID NO. 81.

Embodiment D60 the multi-chain chimeric polypeptide of any one of embodiments D53 to D59, wherein the sequence of soluble human IL-12α (p 35) comprises a sequence at least 80% identical to SEQ ID NO. 80.

Embodiment D61 the multi-chain chimeric polypeptide of embodiment D60, wherein the sequence of soluble human IL-12α (p 35) comprises a sequence at least 90% identical to SEQ ID NO 80.

Embodiment D62 the multi-chain chimeric polypeptide of embodiment D61, wherein the sequence of soluble human IL-12α (p 35) comprises a sequence at least 95% identical to SEQ ID NO 80.

Embodiment D63 the multi-chain chimeric polypeptide of embodiment D62, wherein the sequence of soluble human IL-12α (p 35) comprises SEQ ID NO 80.

Embodiment D64 the multi-chain chimeric polypeptide of embodiment D1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 174.

Embodiment D65 the multi-chain chimeric polypeptide of embodiment D64, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 174.

Embodiment D66 the multi-chain chimeric polypeptide of embodiment D65, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 174.

Embodiment D67 the multi-chain chimeric polypeptide of embodiment D66, wherein the first chimeric polypeptide comprises SEQ ID NO. 174.

Embodiment D68 the multi-chain chimeric polypeptide of embodiment D67, wherein the first chimeric polypeptide comprises SEQ ID NO. 176.

Embodiment D69 the multi-chain chimeric polypeptide of any one of embodiments D1 and D64 to D68, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 178.

Embodiment D70 the multi-chain chimeric polypeptide of embodiment D69, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 178.

Embodiment D71 the multi-chain chimeric polypeptide of embodiment D70, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 178.

Embodiment D72 the multi-chain chimeric polypeptide of embodiment D71, wherein the second chimeric polypeptide comprises SEQ ID NO. 178.

Embodiment D73 the multi-chain chimeric polypeptide of embodiment D72, wherein the second chimeric polypeptide comprises SEQ ID NO. 180.

Embodiment D74 the multi-chain chimeric polypeptide of any one of embodiments D1 to D63, wherein the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more additional antigen binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment D75. The multi-chain chimeric polypeptide of embodiment D74, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more other antigen binding domains, and/or a linker sequence between at least one of the one or more other antigen binding domains and the first domain in the pair of affinity domains.

Embodiment D76 the multi-chain chimeric polypeptide of any one of embodiments D1 to D63, wherein the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and/or C-terminus of the first chimeric polypeptide.

Embodiment D77. The multi-chain chimeric polypeptide of embodiment D76, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains directly abuts the first domain in the pair of affinity domains.

Embodiment D78. The multi-chain chimeric polypeptide of embodiment D76, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains.

Embodiment D79. The multi-chain chimeric polypeptide of embodiment D76, wherein in the first chimeric polypeptide at least one of the one or more additional target binding domains directly adjoins the first target binding domain.

Embodiment D80. The multi-chain chimeric polypeptide of embodiment D76, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain.

Embodiment D81 the multi-chain chimeric polypeptide of embodiment D76, wherein at least one of the one or more additional target binding domains is disposed N-terminal and/or C-terminal to the first chimeric polypeptide and at least one of the one or more additional target binding domains is located between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment D82. The multi-chain chimeric polypeptide of embodiment D81, wherein in the first chimeric polypeptide, at least one additional target binding domain of the one or more additional target binding domains disposed N-terminally directly abuts the first target binding domain or the first domain of the pair of affinity domains.

Embodiment D83. The multi-chain chimeric polypeptide of embodiment D81, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment D84. The multi-chain chimeric polypeptide of embodiment D81, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed at the C-terminus directly abuts the first target binding domain or the first domain in the pair of affinity domains.

Embodiment D85 the multi-chain chimeric polypeptide of embodiment D81, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment D86 the multi-chain chimeric polypeptide of embodiment D81, wherein at least one of the one or more additional target binding domains located between the soluble tissue factor domain and the first domain of the pair of affinity domains directly adjoins the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment D87 the multi-chain chimeric polypeptide of embodiment D81, wherein the first chimeric polypeptide further comprises a linker sequence disposed as follows: (i) Between the soluble tissue factor domain and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains; and/or (ii) between a first domain of the pair of affinity domains and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains being located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment D88 the multi-chain chimeric polypeptide of any one of embodiments D1 to D63 and D74 to D87, wherein the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment D89 the multi-chain chimeric polypeptide of embodiment D88, wherein in the second chimeric polypeptide at least one of the one or more additional target binding domains directly abuts the second domain in the pair of affinity domains.

Embodiment D90. The multi-chain chimeric polypeptide of embodiment D88, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain in the pair of affinity domains.

Embodiment D91 the multi-chain chimeric polypeptide of embodiment D88, wherein in the second chimeric polypeptide at least one of the one or more additional target binding domains directly adjoins the second target binding domain.

Embodiment d92. The multi-chain chimeric polypeptide of embodiment B88, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

The multi-chain chimeric polypeptide of any one of embodiments D74 to D92, wherein two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to the same antigen.

Embodiment d94. The multi-chain chimeric polypeptide of embodiment B93, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope.

Embodiment d95. The multi-chain chimeric polypeptide of embodiment B94, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

Embodiment D96 the multi-chain chimeric polypeptide of any one of embodiments D74 to D92, wherein the first target binding domain, the second target binding domain and one or more additional target binding domains specifically bind to different antigens.

Embodiment D97 the multi-chain chimeric polypeptide of any one of embodiments D74 to D96, wherein one or more additional antigen binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-beta receptor II (TGF-. Beta.RII), ligands of DNAM-1, ligands of TGF-46, TGF-beta-RIII ligands for NKp44, NKG2D, NKp30, scMHCI, scMHCII, scTCR, IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, stem Cell Factor (SCF), stem cell-like tyrosine kinase 3 (FLT 3L), MICA, MICB, ULP16 binding protein, CD155 and CD 28.

Embodiment D98 the multi-chain chimeric polypeptide of any one of embodiments D74 to D96, wherein one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment d99. The multi-chain chimeric polypeptide of embodiment B98, wherein the soluble interleukin, cytokine or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding proteins.

Embodiment D100 the multi-chain chimeric polypeptide of any one of embodiments D74 to D96, wherein one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment D101 the multi-chain chimeric polypeptide of embodiment B100, wherein the soluble receptor is soluble TGF- β receptor II (TGF- βrii), soluble TGF- βriii, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, or soluble CD28.

Embodiment D102 a composition comprising any one of the multi-chain chimeric polypeptides of embodiments D1 to D101.

Embodiment D103 the composition of embodiment D102, wherein the composition is a pharmaceutical composition.

Embodiment D104 a kit comprising at least one dose of the composition of embodiment D102 or D103.

Embodiment D105 a nucleic acid encoding any one of the multi-chain chimeric polypeptides of any one of embodiments D1 to D101.

Embodiment D106. A vector comprising the nucleic acid of embodiment D105.

Embodiment D107 the vector of embodiment D106, wherein the vector is an expression vector.

Embodiment D108 a cell comprising the nucleic acid of embodiment D105 or the vector of embodiment D106 or D107.

Embodiment d109 a method of producing a multi-chain chimeric polypeptide comprising:

culturing the cells of embodiment D108 in a culture medium under conditions sufficient to cause production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cells and/or culture medium.

Embodiment D110 a multi-chain chimeric polypeptide produced by the method of embodiment D109.

Embodiment D111 the multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO 97.

Embodiment D112 the multi-chain chimeric polypeptide of embodiment D111, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 97.

Embodiment D113 the multi-chain chimeric polypeptide of embodiment D112, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 97.

Embodiment D114 the multi-chain chimeric polypeptide of embodiment D113, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO 97.

Embodiment D115 the multi-chain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 98.

Embodiment D116 the multi-chain chimeric polypeptide of embodiment D115, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 98.

Embodiment D117 the multi-chain chimeric polypeptide of embodiment D116, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 98.

Embodiment D118 the multi-chain chimeric polypeptide of embodiment D117, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO. 98.

Embodiment e1. A multi-chain chimeric polypeptide comprising:

(a) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(b) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of a first domain and a second domain of a pair of affinity domains; and is also provided with

The first target binding domain and the second target binding domain each independently specifically bind to a ligand of the receptor for IL-21 or the tumor growth factor receptor βii (tgfbetarii).

Embodiment E2. The multi-chain chimeric polypeptide of embodiment E1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

Embodiment E3. the multi-chain chimeric polypeptide of embodiment E1, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment E4. the multi-chain chimeric polypeptide of any one of embodiments E1 to E3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

Embodiment E5. the multi-chain chimeric polypeptide of any one of embodiments E1 to E3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment E6. the multi-chain chimeric polypeptide of any one of embodiments E1 to E5, wherein the second domain of a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

Embodiment E7. the multi-chain chimeric polypeptide of any one of embodiments E1 to E5, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

Embodiment E8. the multi-chain chimeric polypeptide of any one of embodiments E1 to E7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment E9. the multi-chain chimeric polypeptide of embodiment E8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment E10. The multi-chain chimeric polypeptide of embodiment E9, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 93.

Embodiment E11. The multi-chain chimeric polypeptide of embodiment E10, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment E12. The multi-chain chimeric polypeptide of any one of embodiments E8 to E11, wherein the soluble human tissue factor domain does not comprise one or more of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment E13. The multi-chain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain does not comprise any of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment E14 the multi-chain chimeric polypeptide of any one of embodiments E1 to E13, wherein the soluble tissue factor domain is incapable of binding to factor vila.

Embodiment E15 the multi-chain chimeric polypeptide of any one of embodiments E1 to E14, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment E16 the multi-chain chimeric polypeptide of any one of embodiments E1 to E15, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment E17 the multi-chain chimeric polypeptide of any one of embodiments E1 to E16, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide.

Embodiment E18 the multi-chain chimeric polypeptide of any one of embodiments E1 to E17, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment E19 the multi-chain chimeric polypeptide of any one of embodiments E1 to E18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment E20 the multi-chain chimeric polypeptide of embodiment E19, wherein the signal sequence comprises SEQ ID NO. 117.

Embodiment E21 the multi-chain chimeric polypeptide of embodiment E20, wherein the signal sequence is SEQ ID NO. 117.

Embodiment E22 the multi-chain chimeric polypeptide of any one of embodiments E1 to E21, wherein the pair of affinity domains is a sushi domain from the human IL-15 receptor alpha chain (IL-15 Rα) and soluble IL-15.

Embodiment E23 the multi-chain chimeric polypeptide of embodiment E22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment E24. The multi-chain chimeric polypeptide of embodiment E22, wherein the soluble IL-15 comprises a sequence at least 80% identical to SEQ ID NO. 82.

Embodiment E25 the multi-chain chimeric polypeptide of embodiment E24, wherein the soluble IL-15 comprises a sequence at least 90% identical to SEQ ID NO. 82.

Embodiment E26. The multi-chain chimeric polypeptide of embodiment E25, wherein the soluble IL-15 comprises a sequence at least 95% identical to SEQ ID NO. 82.

Embodiment E27. The multi-chain chimeric polypeptide of embodiment E26, wherein the soluble IL-15 comprises SEQ ID NO. 82.

Embodiment E28 the multi-chain chimeric polypeptide of any one of embodiments E22 to E27, wherein the sushi domain of IL-15Rα comprises a sushi domain from human IL-15Rα.

Embodiment E29. The multi-chain chimeric polypeptide of embodiment E28, wherein the sushi domain from human IL-15Rα comprises a sequence 80% identical to SEQ ID NO 113.

Embodiment E30. The multi-chain chimeric polypeptide of embodiment E29, wherein the sushi domain from human IL-15Rα comprises a sequence 90% identical to SEQ ID NO 113.

Embodiment E31 the multi-chain chimeric polypeptide of embodiment E30, wherein the sushi domain from human IL-15Rα comprises a sequence 95% identical to SEQ ID NO 113.

Embodiment E32 the multi-chain chimeric polypeptide of embodiment E31, wherein the sushi domain from human IL-15Rα comprises SEQ ID NO 113.

Embodiment E33 the multi-chain chimeric polypeptide of embodiment E28, wherein the sushi domain from human IL-15Rα is mature full-length IL-15Rα.

Embodiment E34 the multi-chain chimeric polypeptide of any one of embodiments E1 to E21, wherein a pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

Embodiment E35 the multi-chain chimeric polypeptide of any one of embodiments E1 to E34, wherein one or both of the first target binding domain and the second target binding domain is an antigen binding domain.

Embodiment E36. The multi-chain chimeric polypeptide of embodiment E35, wherein the first target binding domain and the second target binding domain are antigen binding domains.

Embodiment E37 the multi-chain chimeric polypeptide of embodiment E35 or E36, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment E38 the multi-chain chimeric polypeptide of any one of embodiments E1 to E34, wherein one or both of the first target binding domain and the second target binding domain is soluble IL-21 or soluble tgfbetarii.

Embodiment E39 the multi-chain chimeric polypeptide of any one of embodiments E1 to E38, wherein both the first target binding domain and the second target binding domain specifically bind to a ligand of the receptor for IL-21 or tgfbetarii.

Embodiment E40. The multi-chain chimeric polypeptide of embodiment E39, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment E41 the multi-chain chimeric polypeptide of embodiment E40, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment E42. The multi-chain chimeric polypeptide of any one of embodiments E1 to E38, wherein the first target binding domain specifically binds to a ligand of tgfbetarii and the second target binding domain specifically binds to a receptor of IL-21.

Embodiment E43 the multi-chain chimeric polypeptide of any one of embodiments E1 to E38, wherein the first target binding domain specifically binds to a receptor for IL-21 and the second target binding domain specifically binds to a ligand for tgfβrii.

Embodiment E44 the multi-chain chimeric polypeptide of embodiment E43, wherein the first target binding domain comprises soluble IL-21.

Embodiment E45 the multi-chain chimeric polypeptide of embodiment E44, wherein the soluble IL-21 is soluble human IL-21.

Embodiment E46. The multi-chain chimeric polypeptide of embodiment E45, wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID NO 83.

Embodiment E47. The multi-chain chimeric polypeptide of embodiment E46, wherein the soluble human IL-21 comprises a sequence that is at least 90% identical to SEQ ID NO. 83.

Embodiment E48 the multi-chain chimeric polypeptide of embodiment E47, wherein the soluble human IL-21 comprises a sequence at least 95% identical to SEQ ID NO 83.

Embodiment E49 the multi-chain chimeric polypeptide of embodiment E48, wherein the soluble human IL-21 comprises the sequence of SEQ ID NO. 83.

Embodiment E50. The multi-chain chimeric polypeptide of any one of embodiments E43 to E49, wherein the second target binding domain comprises soluble tgfbetarii.

Embodiment E51 the multi-chain chimeric polypeptide of embodiment E50, wherein the soluble tgfbetarii is a soluble human tgfbetarii.

Embodiment E52 the multi-chain chimeric polypeptide of embodiment E51, wherein the soluble human tgfbetarii comprises a first sequence of soluble human tgfbetarii and a second sequence of soluble human tgfbetarii.

Embodiment E53 the multi-chain chimeric polypeptide of embodiment E52, wherein the soluble human tgfbetarii further comprises a linker sequence between the first sequence of the soluble tgfbetarii class and the second sequence of the soluble human tgfbetarii.

Embodiment E54. The multi-chain chimeric polypeptide of embodiment E53, wherein the linker sequence comprises SEQ ID NO. 102.

Embodiment E55. The multi-chain chimeric polypeptide of any one of embodiments E52 to E54, wherein the first sequence of soluble human tgfbetarii comprises a sequence at least 80% identical to SEQ ID No. 183.

Embodiment E56 the multi-chain chimeric polypeptide of embodiment E55, wherein the first sequence of soluble human TGF-beta RII comprises a sequence at least 90% identical to SEQ ID NO 183.

Embodiment E57. The multi-chain chimeric polypeptide of embodiment E56, wherein the first sequence of soluble human TGF-beta RII comprises a sequence at least 95% identical to SEQ ID NO 183.

Embodiment E58 the multi-chain chimeric polypeptide of embodiment E57, wherein the first sequence of soluble human TGF-beta RII comprises SEQ ID NO 183.

Embodiment E59 the multi-chain chimeric polypeptide of any one of embodiments E52 to E58, wherein the second sequence of soluble human tgfbetarii comprises a sequence at least 80% identical to SEQ ID No. 184.

Embodiment E60. The multi-chain chimeric polypeptide of embodiment E59, wherein the second sequence of soluble human tgfbetarii comprises a sequence at least 90% identical to SEQ ID No. 184.

Embodiment E61 the multi-chain chimeric polypeptide of embodiment E60, wherein the second sequence of soluble human TGF-beta RII comprises a sequence at least 95% identical to SEQ ID NO 184.

Embodiment E62. The multi-chain chimeric polypeptide of embodiment E61, wherein the second sequence of soluble human TGF-beta RII comprises SEQ ID NO:184.

Embodiment E63 the multi-chain chimeric polypeptide of embodiment E1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 189.

Embodiment E64. The multi-chain chimeric polypeptide of embodiment E63, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 189.

Embodiment E65. The multi-chain chimeric polypeptide of embodiment E64, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 189.

Embodiment E66. The multi-chain chimeric polypeptide of embodiment E65, wherein the first chimeric polypeptide comprises SEQ ID NO. 189.

Embodiment E67 the multi-chain chimeric polypeptide of embodiment E66, wherein the first chimeric polypeptide comprises SEQ ID NO. 191.

Embodiment E68 the multi-chain chimeric polypeptide of any one of embodiments E1 and E63 to E67, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO 193.

Embodiment E69. the multi-chain chimeric polypeptide of embodiment E68, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 193.

Embodiment E70. The multi-chain chimeric polypeptide of embodiment E69, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 193.

Embodiment E71 the multi-chain chimeric polypeptide of embodiment E70, wherein the second chimeric polypeptide comprises SEQ ID NO. 193.

Embodiment E72 the multi-chain chimeric polypeptide of embodiment E71, wherein the second chimeric polypeptide comprises SEQ ID NO. 195.

Embodiment E73. The multi-chain chimeric polypeptide of any one of embodiments E1 to E62, wherein the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more additional antigen binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment E74 the multi-chain chimeric polypeptide of embodiment E73, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more other antigen binding domains, and/or a linker sequence between at least one of the one or more other antigen binding domains and the first domain in the pair of affinity domains.

Embodiment E75. The multi-chain chimeric polypeptide of any one of embodiments E1 to E62, wherein the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and/or C-terminus of the first chimeric polypeptide.

Embodiment E76. The multi-chain chimeric polypeptide of embodiment E75, wherein in the first chimeric polypeptide at least one of the one or more additional target binding domains directly abuts the first domain in the pair of affinity domains.

Embodiment E77 the multi-chain chimeric polypeptide of embodiment E75, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains.

Embodiment E78. The multi-chain chimeric polypeptide of embodiment E75, wherein in the first chimeric polypeptide at least one of the one or more additional target binding domains directly adjoins the first target binding domain.

Embodiment E79 the multi-chain chimeric polypeptide of embodiment E75, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain.

Embodiment E80. The multi-chain chimeric polypeptide of embodiment E75, wherein at least one of the one or more additional target binding domains is disposed N-terminal and/or C-terminal to the first chimeric polypeptide, and at least one of the one or more additional target binding domains is located between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment E81. The multi-chain chimeric polypeptide of embodiment E80, wherein in the first chimeric polypeptide, at least one additional target binding domain of the one or more additional target binding domains disposed N-terminally directly abuts the first target binding domain or the first domain of the pair of affinity domains.

Embodiment E82. The multi-chain chimeric polypeptide of embodiment E80, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment E83. The multi-chain chimeric polypeptide of embodiment E80, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed at the C-terminus directly abuts the first target binding domain or the first domain of the pair of affinity domains.

Embodiment E84 the multi-chain chimeric polypeptide of embodiment E80, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment E85 the multi-chain chimeric polypeptide of embodiment E80, wherein at least one of the one or more additional target binding domains located between the soluble tissue factor domain and the first domain of the pair of affinity domains directly adjoins the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment E86 the multi-chain chimeric polypeptide of embodiment E80, wherein the first chimeric polypeptide further comprises a linker sequence disposed as follows: (i) Between the soluble tissue factor domain and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains; and/or (ii) between a first domain of the pair of affinity domains and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains being located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment E87 the multi-chain chimeric polypeptide of any one of embodiments E1 to E62 and E73 to E86, wherein the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment E88. The multi-chain chimeric polypeptide of embodiment E87, wherein in the second chimeric polypeptide, at least one of the one or more additional target binding domains directly abuts the second domain in the pair of affinity domains.

Embodiment E89 the multi-chain chimeric polypeptide of embodiment E87, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain in the pair of affinity domains.

Embodiment E90. The multi-chain chimeric polypeptide of embodiment E87, wherein in the second chimeric polypeptide, at least one of the one or more additional target binding domains directly adjoins the second target binding domain.

Embodiment E91 the multi-chain chimeric polypeptide of embodiment E87, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

Embodiment E92. The multi-chain chimeric polypeptide of any one of embodiments E73 to E91, wherein two or more of the first target binding domain, the second target binding domain and one or more additional target binding domains specifically bind to the same antigen.

Embodiment E93 the multi-chain chimeric polypeptide of embodiment E92, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope.

Embodiment E94. The multi-chain chimeric polypeptide of embodiment E93, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

Embodiment E95 the multi-chain chimeric polypeptide of any one of embodiments E73 to E91, wherein the first target binding domain, the second target binding domain and one or more additional target binding domains specifically bind to different antigens.

Embodiment E96. The multi-chain chimeric polypeptide of any one of embodiments E73 to E95, wherein one or more additional antigen binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, ligands for PDGF-D, TGF-beta receptor II (TGF-. Beta.RII), ligands for TGF-. Beta.RIII, ligands for DNAM-1, ligands for NKp46 ligands for NKp44, NKG2D, NKp30, scMHCI, scMHCII, scTCR, IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, stem Cell Factor (SCF), stem cell-like tyrosine kinase 3 (FLT 3L), MICA, MICB, ULP16 binding protein, CD155 and CD 28.

Embodiment E97 the multi-chain chimeric polypeptide of any one of embodiments E73 to E95, wherein one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment E98 the multi-chain chimeric polypeptide of embodiment E97, wherein the soluble interleukin, cytokine or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding proteins.

Embodiment E99 the multi-chain chimeric polypeptide of any one of embodiments E73 to E95, wherein one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment E100 the multi-chain chimeric polypeptide of embodiment E99, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155 or soluble CD28.

Embodiment E101. A composition comprising any of the multiple chain chimeric polypeptides of embodiments E1 to E100.

Embodiment E102 the composition of embodiment E101, wherein the composition is a pharmaceutical composition.

Embodiment E103 a kit comprising at least one dose of the composition of embodiment E101 or E102.

Embodiment E104. A nucleic acid encoding any one of the multi-chain chimeric polypeptides of any one of embodiments E1 to E100.

Embodiment E105. A vector comprising the nucleic acid of embodiment E104.

Embodiment E106 the vector of embodiment E105, wherein the vector is an expression vector.

Embodiment E107 a cell comprising the nucleic acid of embodiment C104 or the vector of embodiment E105 or E106.

Embodiment e108. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cells of embodiment E107 in a culture medium under conditions sufficient to cause production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cells and/or culture medium.

Embodiment E109. A multi-chain chimeric polypeptide produced by the method of embodiment E108.

Embodiment E110. The multi-chain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO 97.

Embodiment E111 the multi-chain chimeric polypeptide of embodiment E110, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO 97.

Embodiment E112 the multi-chain chimeric polypeptide of embodiment E111, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 97.

Embodiment E113 the multi-chain chimeric polypeptide of embodiment E112, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO 97.

Embodiment E114. The multi-chain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 98.

Embodiment E115 the multi-chain chimeric polypeptide of embodiment E114, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 98.

Embodiment E116 the multi-chain chimeric polypeptide of embodiment E115, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 98.

Embodiment E117 the multi-chain chimeric polypeptide of embodiment E116, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO. 98.

Embodiment f1. A multi-chain chimeric polypeptide comprising:

(c) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(d) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of a first domain and a second domain of a pair of affinity domains;

the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-21 or IL-7.

Embodiment F2. the multi-chain chimeric polypeptide of embodiment F1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

Embodiment F3. the multi-chain chimeric polypeptide of embodiment F1, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment F4. the multi-chain chimeric polypeptide of any one of embodiments F1 to F3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

Embodiment F5. the multi-chain chimeric polypeptide of any one of embodiments F1 to F3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment F6. the multi-chain chimeric polypeptide of any one of embodiments F1 to F5, wherein the second domain of a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

Embodiment F7. the multi-chain chimeric polypeptide of any one of embodiments F1 to F5, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

Embodiment F8. the multi-chain chimeric polypeptide of any one of embodiments F1 to F7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment F9. the multi-chain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment F10. The multi-chain chimeric polypeptide of embodiment F9, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 93.

Embodiment F11. The multi-chain chimeric polypeptide of embodiment F10, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment F12 the multi-chain chimeric polypeptide of embodiment F11, wherein the soluble human tissue factor domain comprises SEQ ID NO. 93.

Embodiment F13. The multi-chain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 97.

Embodiment F14. The multi-chain chimeric polypeptide of embodiment F13, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 97.

Embodiment F15 the multi-chain chimeric polypeptide of embodiment F14, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID No. 97.

Embodiment F16. The multi-chain chimeric polypeptide of embodiment F11, wherein the soluble human tissue factor domain comprises SEQ ID NO. 97.

Embodiment F17 the multi-chain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 98.

Embodiment F18. The multi-chain chimeric polypeptide of embodiment F17, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 98.

Embodiment F19. The multi-chain chimeric polypeptide of embodiment F18, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 98.

Embodiment F20. The multi-chain chimeric polypeptide of embodiment F11, wherein the soluble human tissue factor domain comprises SEQ ID NO. 98.

Embodiment F21. The multi-chain chimeric polypeptide of any one of embodiments F8 to F11, F13 to F15, and F17 to F19, wherein the soluble human tissue factor domain does not comprise one or more of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment F22. The multi-chain chimeric polypeptide of embodiment F21, wherein the soluble human tissue factor domain does not comprise any of the following:

Lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment F23 the multi-chain chimeric polypeptide of any one of embodiments F1 to F22, wherein the soluble tissue factor domain is incapable of binding to factor vila.

Embodiment F24 the multi-chain chimeric polypeptide of any one of embodiments F1 to F23, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment F25 the multi-chain chimeric polypeptide of any one of embodiments F1 to F24, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment F26 the multi-chain chimeric polypeptide of any one of embodiments F1 to F25, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide.

Embodiment F27 the multi-chain chimeric polypeptide of any one of embodiments F1 to F26, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment F28 the multi-chain chimeric polypeptide of any one of embodiments F1 to F27, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment F29. The multi-chain chimeric polypeptide of embodiment F28, wherein the signal sequence comprises SEQ ID NO. 117.

Embodiment F30 the multi-chain chimeric polypeptide of embodiment F28, wherein the signal sequence is SEQ ID NO. 328.

Embodiment F31 the multi-chain chimeric polypeptide of any one of embodiments F1 to F30, wherein the pair of affinity domains is a sushi domain from the human IL-15 receptor alpha chain (IL-15 rα) and soluble IL-15.

Embodiment F32 the multi-chain chimeric polypeptide of embodiment F31, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment F33 the multi-chain chimeric polypeptide of embodiment F31, wherein the soluble IL-15 comprises a sequence at least 80% identical to SEQ ID NO.

Embodiment F34 the multi-chain chimeric polypeptide of embodiment F31, wherein the soluble IL-15 comprises a sequence at least 80% identical to SEQ ID NO.

Embodiment F35 the multi-chain chimeric polypeptide of embodiment F31, wherein the soluble IL-15 comprises a sequence at least 80% identical to SEQ ID NO.

Embodiment F36 the multi-chain chimeric polypeptide of embodiment F35, wherein the soluble IL-15 comprises SEQ ID NO. 82.

Embodiment F37 the multi-chain chimeric polypeptide of any one of embodiments F31 to F36, wherein the sushi domain of IL-15 ra comprises a sushi domain from human IL-15 ra.

Embodiment F38. The multi-chain chimeric polypeptide of embodiment F37, wherein the sushi domain from human IL-15Rα comprises a sequence at least 80% identical to SEQ ID NO 113.

Embodiment F39. The multi-chain chimeric polypeptide of embodiment F38, wherein the sushi domain from human IL-15Rα comprises a sequence at least 90% identical to SEQ ID NO 113.

Embodiment F40. The multi-chain chimeric polypeptide of embodiment F39, wherein the sushi domain from human IL-15Rα comprises a sequence at least 95% identical to SEQ ID NO 113.

Embodiment F41 the multi-chain chimeric polypeptide of embodiment F40, wherein the sushi domain from human IL-15Rα comprises SEQ ID NO 113.

Embodiment F42 the multi-chain chimeric polypeptide of embodiment F37, wherein the sushi domain from human IL-15Rα is mature full-length IL-15Rα.

Embodiment F43 the multi-chain chimeric polypeptide of any one of embodiments F1 to F30, wherein a pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

Embodiment F44 the multi-chain chimeric polypeptide of any one of embodiments F1 to F43, wherein one or both of the first target binding domain and the second target binding domain is an agonistic antigen binding domain.

Embodiment F45 the multi-chain chimeric polypeptide of embodiment F44, wherein the first target binding domain and the second target binding domain are each an agonistic antigen binding domain.

Embodiment F46 the multi-chain chimeric polypeptide of embodiment F44 or F45, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment F47 the multi-chain chimeric polypeptide of any one of embodiments F1 to F43, wherein one or both of the first target binding domain and the second target binding domain is soluble IL-21 or soluble IL-7.

Embodiment F48 the multi-chain chimeric polypeptide of embodiment F47, wherein the first target binding domain and the second target binding domain are each independently soluble IL-21 or soluble IL-7.

Embodiment F49 the multi-chain chimeric polypeptide of any one of embodiments F1 to F48, wherein both the first target binding domain and the second target binding domain specifically bind to a receptor for IL-21 or IL-7.

Embodiment F50 the multi-chain chimeric polypeptide of embodiment F49, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment F51 the multi-chain chimeric polypeptide of embodiment F50, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment F52 the multi-chain chimeric polypeptide of any one of embodiments F1 to F48, wherein the first target binding domain specifically binds to a receptor for IL-21 and the second target binding domain specifically binds to a receptor for IL-7.

Embodiment F53 the multi-chain chimeric polypeptide of any one of embodiments F1 to F48, wherein the first target binding domain specifically binds to a receptor for IL-7 and the second target binding domain specifically binds to a receptor for IL-21.

Embodiment F54. The multi-chain chimeric polypeptide of embodiment F53, wherein the first target binding domain comprises soluble IL-21.

Embodiment F55 the multi-chain chimeric polypeptide of embodiment F54, wherein the soluble IL-21 is soluble human IL-21.

Embodiment F56 the multi-chain chimeric polypeptide of embodiment F55, wherein the soluble human IL-21 comprises a sequence at least 80% identical to SEQ ID NO 83.

Embodiment F57 the multi-chain chimeric polypeptide of embodiment F56, wherein the soluble human IL-21 comprises a sequence at least 90% identical to SEQ ID NO 83.

Embodiment F58 the multi-chain chimeric polypeptide of embodiment F57, wherein the soluble human IL-21 comprises a sequence that is at least 95% identical to SEQ ID NO. 83.

Embodiment F59 the multi-chain chimeric polypeptide of embodiment F58, wherein the soluble human IL-21 comprises the sequence of SEQ ID NO. 83.

Embodiment F60 the multi-chain chimeric polypeptide of any one of embodiments F53 to F59, wherein the second target binding domain comprises soluble IL-7.

Embodiment F61 the multi-chain chimeric polypeptide of embodiment D60, wherein the soluble IL-7 is soluble human IL-7.

Embodiment F62. The multi-chain chimeric polypeptide of embodiment F61, wherein the soluble human IL-7 comprises a sequence at least 80% identical to SEQ ID NO. 79.

Embodiment F63. The multi-chain chimeric polypeptide of embodiment F62, wherein the soluble human IL-7 comprises a sequence that is at least 90% identical to SEQ ID NO. 79.

Embodiment F64 the multi-chain chimeric polypeptide of embodiment F63, wherein the soluble human IL-7 comprises a sequence at least 95% identical to SEQ ID NO 79.

Embodiment F65. The multi-chain chimeric polypeptide of embodiment F64, wherein the soluble human IL-7 comprises the sequence of SEQ ID NO. 79.

Embodiment F66. The multi-chain chimeric polypeptide of embodiment F1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 207.

Embodiment F67 the multi-chain chimeric polypeptide of embodiment F66, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 207.

Embodiment F68. The multi-chain chimeric polypeptide of embodiment F67, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 207.

Embodiment F69. The multi-chain chimeric polypeptide of embodiment F68, wherein the first chimeric polypeptide comprises SEQ ID NO. 207.

Embodiment F70. The multi-chain chimeric polypeptide of embodiment F69, wherein the first chimeric polypeptide comprises SEQ ID NO. 209.

Embodiment F71 the multi-chain chimeric polypeptide of any one of embodiments F1 and F66 to F70, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 211.

Embodiment F72 the multi-chain chimeric polypeptide of embodiment F71, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 211.

Embodiment F73 the multi-chain chimeric polypeptide of embodiment F72, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 211.

Embodiment F74 the multi-chain chimeric polypeptide of embodiment F73, wherein the second chimeric polypeptide comprises SEQ ID NO. 211.

Embodiment F75 the multi-chain chimeric polypeptide of embodiment F74, wherein the second chimeric polypeptide comprises SEQ ID NO:213.

Embodiment F76 the multi-chain chimeric polypeptide of embodiment F1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO 199.

Embodiment F77 the multi-chain chimeric polypeptide of embodiment F76, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO 199.

Embodiment F78. The multi-chain chimeric polypeptide of embodiment F77, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO 199.

Embodiment F79 the multi-chain chimeric polypeptide of embodiment F68, wherein the first chimeric polypeptide comprises SEQ ID NO 199.

Embodiment F80 the multi-chain chimeric polypeptide of embodiment F69, wherein the first chimeric polypeptide comprises SEQ ID NO. 201.

Embodiment F81 the multi-chain chimeric polypeptide of any one of embodiments F1 and F76 to F80, wherein the second chimeric polypeptide comprises a sequence that is less than 80% identical to SEQ ID No. 203.

Embodiment F82 the multi-chain chimeric polypeptide of embodiment F81, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 203.

Embodiment F83 the multi-chain chimeric polypeptide of embodiment F82, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 203.

Embodiment F84 the multi-chain chimeric polypeptide of embodiment F83, wherein the second chimeric polypeptide comprises SEQ ID NO. 203.

Embodiment F85 the multi-chain chimeric polypeptide of embodiment F84, wherein the second chimeric polypeptide comprises SEQ ID NO. 209.

The multiplex chimeric polypeptide of any one of embodiments F1 to F65, wherein the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more additional antigen binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment F87. The multi-chain chimeric polypeptide of embodiment F86, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more other antigen binding domains, and/or a linker sequence between at least one of the one or more other antigen binding domains and the first domain in the pair of affinity domains.

Embodiment F88 the multi-chain chimeric polypeptide of any one of embodiments F1 to F65, wherein the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and/or C-terminus of the first chimeric polypeptide.

Embodiment F89 the multi-chain chimeric polypeptide of embodiment F88, wherein in the first chimeric polypeptide at least one of the one or more additional target binding domains directly abuts the first domain in the pair of affinity domains.

Embodiment F90. The multi-chain chimeric polypeptide of embodiment F88, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains.

Embodiment F91 the multi-chain chimeric polypeptide of embodiment F88, wherein in the first chimeric polypeptide at least one of the one or more additional target binding domains directly adjoins the first target binding domain.

Embodiment F92. The multi-chain chimeric polypeptide of embodiment F88, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain.

Embodiment F93. The multi-chain chimeric polypeptide of embodiment F88, wherein at least one of the one or more additional target binding domains is disposed N-terminal and/or C-terminal to the first chimeric polypeptide, and at least one of the one or more additional target binding domains is located between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment F94. The multi-chain chimeric polypeptide of embodiment F93, wherein in the first chimeric polypeptide, at least one additional target binding domain of the one or more additional target binding domains disposed N-terminally directly abuts the first target binding domain or the first domain of the pair of affinity domains.

Embodiment F95. The multi-chain chimeric polypeptide of embodiment F93, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment F96. The multi-chain chimeric polypeptide of embodiment F93, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed at the C-terminus directly abuts the first target binding domain or the first domain of the pair of affinity domains.

Embodiment F97 the multi-chain chimeric polypeptide of embodiment F93, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment F98. The multi-chain chimeric polypeptide of embodiment F93, wherein at least one of the one or more additional target binding domains located between the soluble tissue factor domain and the first domain of the pair of affinity domains directly abuts the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment F99. The multi-chain chimeric polypeptide of embodiment F93, wherein the first chimeric polypeptide further comprises a linker sequence disposed as follows: (i) Between the soluble tissue factor domain and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains; and/or (ii) between a first domain of the pair of affinity domains and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains being located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment F100 the multi-chain chimeric polypeptide of any one of embodiments F1 to F65 and F86 to F99, wherein the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment F101. The multi-chain chimeric polypeptide of embodiment F100, wherein in the second chimeric polypeptide at least one of the one or more additional target binding domains directly adjoins the second domain in the pair of affinity domains.

Embodiment F102. The multi-chain chimeric polypeptide of embodiment F100, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain in the pair of affinity domains.

Embodiment F103. The multi-chain chimeric polypeptide of embodiment F100, wherein in the second chimeric polypeptide at least one of the one or more additional target binding domains directly adjoins the second target binding domain.

Embodiment F104. The multi-chain chimeric polypeptide of embodiment F100, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

Embodiment F105 the multi-chain chimeric polypeptide of any one of embodiments F86 to F104, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen.

Embodiment F106. The multi-chain chimeric polypeptide of embodiment F105, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope.

Embodiment F107 the multi-chain chimeric polypeptide of embodiment F106, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

Embodiment F108 the multi-chain chimeric polypeptide of any one of embodiments F86 to F104, wherein the first target binding domain, the second target binding domain and one or more additional target binding domains specifically bind to different antigens.

Embodiment F109. The multi-chain chimeric polypeptide of any one of embodiments F86 to F108, wherein one or more additional antigen binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFa, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands of TGF-beta receptor II (TGF-. Beta.RII), ligands of DNAM-1, ligands of TGF-46, TGF-beta-RIII ligands for NKp44, NKG2D, NKp30, scMHCI, scMHCII, scTCR, IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, stem Cell Factor (SCF), stem cell-like tyrosine kinase 3 (FLT 3L), MICA, MICB, ULP16 binding protein, CD155 and CD 28.

Embodiment F110 the multi-chain chimeric polypeptide of any one of embodiments F86 to F108, wherein one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment F111 the multi-chain chimeric polypeptide of embodiment F110, wherein the soluble interleukin, cytokine or ligand protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding proteins.

Embodiment F112 the multi-chain chimeric polypeptide of any one of embodiments F86 to F108, wherein one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment F113 the multi-chain chimeric polypeptide of embodiment F112, wherein the soluble receptor is soluble TGF-beta receptor II (TGF- βrii), soluble TGF- βriii, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, or soluble CD28.

Embodiment F114 a composition comprising any of the multiple chain chimeric polypeptides of embodiments F1 to F113.

Embodiment F115 the composition of embodiment F114 wherein the composition is a pharmaceutical composition.

Embodiment F116 a kit comprising at least one dose of the composition of embodiment F114 or F115.

Embodiment F117 a nucleic acid encoding any one of the multi-chain chimeric polypeptides of any one of embodiments F1 to F113.

Embodiment F118 a vector comprising the nucleic acid of embodiment F117.

Embodiment F119 the vector of embodiment F118, wherein the vector is an expression vector.

Embodiment F120 a cell comprising the nucleic acid of embodiment F117 or the vector of embodiment F118 or F119.

Embodiment f121 a method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cells of embodiment F120 in a culture medium under conditions sufficient to cause production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cells and/or culture medium.

Embodiment F122 a multi-chain chimeric polypeptide produced by the method of embodiment F121.

Embodiment G1. a multi-chain chimeric polypeptide comprising:

(e) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(f) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein:

the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of a first domain and a second domain of a pair of affinity domains; and is also provided with

The first target binding domain and the second target binding domain each independently specifically bind to: IL-7 receptor, CD16, IL-21 receptor, TGF-beta or CD137L receptor.

Embodiment G2. the multi-chain chimeric polypeptide of embodiment G1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

Embodiment G3. the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment G4. the multi-chain chimeric polypeptide of any one of embodiments G1 to G3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

Embodiment G5. the multi-chain chimeric polypeptide of any one of embodiments G1 to G3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment G6. the multi-chain chimeric polypeptide of any one of embodiments G1 to G5, wherein the second domain of a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

Embodiment G7. the multi-chain chimeric polypeptide of any one of embodiments G1 to G5, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

Embodiment G8. the multi-chain chimeric polypeptide of any one of embodiments G1 to G7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment G9. the multi-chain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment G10. The multi-chain chimeric polypeptide of embodiment G9, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 93.

Embodiment G11. The multi-chain chimeric polypeptide of embodiment G10, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment G12. The multi-chain chimeric polypeptide of any one of embodiments G8 to G11, wherein the soluble human tissue factor domain does not comprise one or more of:

Lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment G13. The multi-chain chimeric polypeptide of embodiment G12, wherein the soluble human tissue factor domain does not comprise any of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

Tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment G14 the multi-chain chimeric polypeptide of any one of embodiments G1 to G13, wherein the soluble tissue factor domain is incapable of binding to factor vila.

Embodiment G15 the multi-chain chimeric polypeptide of any one of embodiments G1 to G14, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment G16 the multi-chain chimeric polypeptide of any one of embodiments G1 to G15, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment G17 the multi-chain chimeric polypeptide of any one of embodiments G1 to G16, wherein the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide.

Embodiment G18 the multi-chain chimeric polypeptide of any one of embodiments G1 to G17, wherein the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment G19 the multi-chain chimeric polypeptide of any one of embodiments G1 to G18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment G20 the multi-chain chimeric polypeptide of embodiment G19, wherein the signal sequence comprises SEQ ID NO. 117.

Embodiment G21 the multi-chain chimeric polypeptide of embodiment G20, wherein the signal sequence is SEQ ID NO. 117.

Embodiment G22 the multi-chain chimeric polypeptide of any one of embodiments G1 to G21, wherein the pair of affinity domains is a sushi domain from the human IL-15 receptor alpha chain (IL-15 rα) and soluble IL-15.

Embodiment G23 the multi-chain chimeric polypeptide of embodiment G22, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment G24. The multi-chain chimeric polypeptide of embodiment G22, wherein the soluble IL-15 comprises a sequence at least 80% identical to SEQ ID NO. 82.

Embodiment G25 the multi-chain chimeric polypeptide of embodiment G24, wherein the soluble IL-15 comprises a sequence at least 90% identical to SEQ ID NO. 82.

Embodiment G26 the multi-chain chimeric polypeptide of embodiment G25, wherein the soluble IL-15 comprises a sequence at least 95% identical to SEQ ID NO. 82.

Embodiment G27 the multi-chain chimeric polypeptide of embodiment G26, wherein the soluble IL-15 comprises SEQ ID NO. 82.

Embodiment G28 the multi-chain chimeric polypeptide of any one of embodiments G22 to G27, wherein the sushi domain of IL-15 ra comprises a sushi domain from human IL-15 ra.

Embodiment G29. The multi-chain chimeric polypeptide of embodiment G28, wherein the sushi domain from human IL-15Rα comprises a sequence 80% identical to SEQ ID NO 113.

Embodiment G30. The multi-chain chimeric polypeptide of embodiment G29, wherein the sushi domain from human IL-15Rα comprises a sequence 90% identical to SEQ ID NO 113.

Embodiment G31 the multi-chain chimeric polypeptide of embodiment G30, wherein the sushi domain from human IL-15Rα comprises a sequence 95% identical to SEQ ID NO 113.

Embodiment G32 the multi-chain chimeric polypeptide of embodiment G31, wherein the sushi domain from human IL-15Rα comprises SEQ ID NO 113.

Embodiment G33 the multi-chain chimeric polypeptide of embodiment G28, wherein the sushi domain from human IL-15Rα is mature full length IL-15Rα.

Embodiment G34 the multi-chain chimeric polypeptide of any one of embodiments G1 to G21, wherein a pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

Embodiment G35 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7, CD16 or IL-21.

Embodiment G36 the multi-chain chimeric polypeptide of embodiment G35, wherein the first target binding domain specifically binds to a receptor for IL-7 and the second target binding domain specifically binds to a receptor for CD16 or IL-21.

Embodiment G37 the multi-chain chimeric polypeptide of embodiment G36, wherein the first target binding domain comprises a soluble IL-7 protein.

Embodiment G38 the multi-chain chimeric polypeptide of embodiment G37, wherein the soluble IL-7 is soluble human IL-7.

Embodiment G39 the multi-chain chimeric polypeptide of any one of embodiments G36 to G38, wherein the second antigen binding domain comprises an antigen binding domain that specifically binds to CD 16.

Embodiment G40. The multi-chain chimeric polypeptide of embodiment G39, wherein the second antigen binding domain comprises an scFv that specifically binds to CD 16.

Embodiment G41 the multi-chain chimeric polypeptide of any one of embodiments G36 to G38, wherein the second antigen binding domain specifically binds to a receptor for IL-21.

Embodiment G42 the multi-chain chimeric polypeptide of embodiment G41, wherein the second antigen binding domain comprises soluble IL-21.

Embodiment G43 the multi-chain chimeric polypeptide of embodiment G42, wherein the soluble IL-21 is soluble human IL-21.

Embodiment G44 the multi-chain chimeric polypeptide of any one of embodiments G36 to G40, wherein the second antigen binding domain further comprises an additional target binding domain that specifically binds to a receptor for IL-21.

Embodiment G45 the multi-chain chimeric polypeptide of embodiment G44, wherein the additional target binding domain comprises soluble IL-21.

Embodiment G46 the multi-chain chimeric polypeptide of embodiment G45, wherein the soluble IL-21 is soluble human IL-12.

Embodiment G47 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a receptor for TGF- β, CD16 or IL-21.

Embodiment G48 the multi-chain chimeric polypeptide of embodiment G47, wherein the first target binding domain specifically binds to TGF-beta and the second target binding domain specifically binds to a receptor for CD16 or IL-21.

Embodiment G49 the multispecific chimeric polypeptide of embodiment G48, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G50 the multispecific chimeric polypeptide of embodiment G49, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G51 the multispecific chimeric polypeptide of any one of embodiments G48 to G50, wherein the second target binding domain specifically binds to CD16.

Embodiment G52 the multispecific chimeric polypeptide of embodiment G51, wherein the second antigen-binding domain comprises an antigen-binding domain that specifically binds to CD16.

Embodiment G53 the multi-chain chimeric polypeptide of embodiment G52, wherein the second antigen binding domain comprises an scFv that specifically binds to CD16.

Embodiment G54 the multi-chain chimeric polypeptide of any one of embodiments G48 to G50, wherein the second target binding domain specifically binds to a receptor for IL-21.

Embodiment G55 the multi-chain chimeric polypeptide of embodiment G54, wherein the second target binding domain comprises soluble IL-21.

Embodiment G56. The multi-chain chimeric polypeptide of embodiment G55, wherein the second target binding domain comprises soluble human IL-21.

Embodiment G57 the multi-chain chimeric polypeptide of any one of embodiments G48 to G53, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to a receptor for IL-21.

Embodiment G58 the multi-chain chimeric polypeptide of embodiment G57, wherein the additional target binding domain comprises soluble IL-21.

Embodiment G59 the multi-chain chimeric polypeptide of embodiment G58, wherein the soluble IL-21 is soluble human IL-21.

Embodiment G60 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7.

Embodiment G61 the multi-chain chimeric polypeptide of embodiment G60, wherein the first target binding domain and the second target binding domain comprise soluble IL-7.

Embodiment G62 the multi-chain chimeric polypeptide of embodiment G61, wherein the soluble IL-7 is soluble human IL-7.

Embodiment G63 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to TGF- β.

Embodiment G64 the multispecific chimeric polypeptide of embodiment G63, wherein the first target binding domain and the second target binding domain are soluble TGF- β receptors.

Embodiment G65 the multispecific chimeric polypeptide of embodiment G64, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G66. The multispecific chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7, a receptor for IL-21, or a receptor for CD 137L.

Embodiment G67 the multi-chain chimeric polypeptide of embodiment G66, wherein the first target binding domain specifically binds to a receptor for IL-7 and the second target binding domain specifically binds to a receptor for IL-21 or a receptor for CD 137L.

Embodiment G68 the multispecific chimeric polypeptide of embodiment G67, wherein the first target binding domain is soluble IL-7.

Embodiment G69 the multispecific chimeric polypeptide of embodiment G68, wherein the soluble IL-7 is soluble human IL-7.

Embodiment G70 the multi-chain chimeric polypeptide of any one of embodiments G67 to G69, wherein the second target binding domain specifically binds to a receptor for IL-21.

Embodiment G71 the multi-chain chimeric polypeptide of embodiment G70, wherein the second target binding domain is soluble IL-21.

Embodiment G72 the multi-chain chimeric polypeptide of embodiment G71, wherein the soluble IL-21 is soluble human IL-21.

Embodiment G73 the multi-chain chimeric polypeptide of any one of embodiments G67 to G69, wherein the second antigen binding domain specifically binds to a receptor of CD137L.

Embodiment G74 the multi-chain chimeric polypeptide of embodiment G73, wherein the second antigen binding domain is soluble CD137L.

Embodiment G75 the multi-chain chimeric polypeptide of embodiment G74, wherein the soluble CD137L is soluble human CD137L.

Embodiment G76 the multi-chain chimeric polypeptide of any one of embodiments G67 to G72, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to a receptor of CD137L.

Embodiment G77 the multi-chain chimeric polypeptide of embodiment G76, wherein the additional target binding domain comprises soluble CD137L.

Embodiment G78 the multi-chain chimeric polypeptide of embodiment G77, wherein the soluble CD137L is soluble human CD137L.

Embodiment G79 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a receptor for IL-7 or TGF- β.

Embodiment G80. The multi-chain chimeric polypeptide of embodiment G79, wherein the first target binding domain specifically binds to a receptor for IL-7 and the second target binding domain specifically binds to TGF- β.

Embodiment G81 the multi-chain chimeric polypeptide of embodiment G80, wherein the first target binding domain comprises a soluble IL-7 protein.

Embodiment G82 the multi-chain chimeric polypeptide of embodiment G81, wherein the soluble IL-7 is soluble human IL-7.

Embodiment G83 the multi-chain chimeric polypeptide of any one of embodiments G80 to G82, wherein the second antigen binding domain comprises an antigen binding domain that specifically binds to TGF- β.

Embodiment G84 the multispecific chimeric polypeptide of embodiment G83, wherein the second target binding domain is a soluble TGF- β receptor.

Embodiment G85 the multispecific chimeric polypeptide of embodiment G84, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G86 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to TGF- β, a receptor for IL-21, or a receptor for CD 137L.

Embodiment G87 the multi-chain chimeric polypeptide of embodiment G86, wherein the first target binding domain specifically binds to TGF- β and the second target binding domain specifically binds to the receptor for IL-21 or the receptor for CD 137L.

Embodiment G88 the multispecific chimeric polypeptide of embodiment G87, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G89 the multispecific chimeric polypeptide of embodiment G88, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G90 the multispecific chimeric polypeptide of any one of embodiments G87 to G89, wherein the second target binding domain specifically binds to a receptor for IL-21.

Embodiment G91 the multi-chain chimeric polypeptide of embodiment G90, wherein the second target binding domain comprises soluble IL-21.

Embodiment G92 the multi-chain chimeric polypeptide of embodiment G91, wherein the second target binding domain comprises soluble human IL-21.

Embodiment G93 the multispecific chimeric polypeptide of any one of embodiments G87 to G89, wherein the second target binding domain specifically binds to a receptor of CD137L.

Embodiment G94 the multi-chain chimeric polypeptide of embodiment G93, wherein the second target binding domain comprises soluble CD137L.

Embodiment G95. The multi-chain chimeric polypeptide of embodiment G94, wherein the second target binding domain comprises soluble human CD137L.

Embodiment G96 the multi-chain chimeric polypeptide of any one of embodiments G87 to G92, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to a receptor of CD137L.

Embodiment G97 the multi-chain chimeric polypeptide of embodiment G96, wherein the additional target binding domain comprises soluble CD137L.

Embodiment G98 the multi-chain chimeric polypeptide of embodiment G97, wherein the soluble CD137L is soluble human CD137L.

Embodiment G99 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a receptor for TGF- β or IL-21.

Embodiment G100. The multi-chain chimeric polypeptide of embodiment G99, wherein the first target binding domain specifically binds to TGF- β and the second target binding domain specifically binds to a receptor for TGF- β or IL-21.

Embodiment G101. The multispecific chimeric polypeptide of embodiment G100, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G102 the multispecific chimeric polypeptide of embodiment G101, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G103 the multispecific chimeric polypeptide of any one of embodiments G100 to G102, wherein the second target binding domain specifically binds to a receptor for IL-21.

Embodiment G104. The multi-chain chimeric polypeptide of embodiment G103, wherein the second target binding domain comprises soluble IL-21.

Embodiment G105 the multi-chain chimeric polypeptide of embodiment G104, wherein the second target binding domain comprises soluble human IL-21.

Embodiment G106 the multispecific chimeric polypeptide of any one of embodiments G100 to G102, wherein the second target binding domain specifically binds to TGF- β.

Embodiment G107 the multispecific chimeric polypeptide of embodiment G106, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G108 the multispecific chimeric polypeptide of embodiment G107, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G109 the multispecific chimeric polypeptide of any one of embodiments G100 to G105, wherein the second polypeptide further comprises an additional target binding domain that specifically binds to TGF- β.

Embodiment G110 the multispecific chimeric polypeptide of embodiment G109, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G111 the multispecific chimeric polypeptide of embodiment G110, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G112 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to TGF- β or IL-16.

Embodiment G113 the multi-chain chimeric polypeptide of embodiment G112, wherein the first target binding domain specifically binds to TGF- β and the second target binding domain specifically binds to TGF- β or IL-16.

Embodiment G114 the multispecific chimeric polypeptide of embodiment G113, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G115 the multispecific chimeric polypeptide of embodiment G114, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G116 the multispecific chimeric polypeptide of any one of embodiments G113 to G115, wherein the second target binding domain specifically binds to IL-16.

Embodiment G117 the multispecific chimeric polypeptide of embodiment G116, wherein the second antigen-binding domain comprises an antigen-binding domain that specifically binds to CD 16.

Embodiment G118 the multi-chain chimeric polypeptide of embodiment G117, wherein the second antigen binding domain comprises an scFv that specifically binds to CD 16.

Embodiment G119 the multispecific chimeric polypeptide of any one of embodiments G113 to G115, wherein the second target binding domain specifically binds to TGF- β.

Embodiment G120 the multispecific chimeric polypeptide of embodiment G119, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G121 the multispecific chimeric polypeptide of embodiment G120, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G122 the multispecific chimeric polypeptide of any one of embodiments G113 to G118, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to TGF- β.

Embodiment G123 the multispecific chimeric polypeptide of embodiment G122, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G124 the multispecific chimeric polypeptide of embodiment G123, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G125 the multi-chain chimeric polypeptide of any one of embodiments G1 to G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a receptor for TGF- β or CD 137L.

Embodiment G126 the multi-chain chimeric polypeptide of embodiment G125, wherein the first target binding domain specifically binds to TGF- β and the second target binding domain specifically binds to a receptor of CD 137L.

Embodiment G127 the multispecific chimeric polypeptide of embodiment G126, wherein the first target binding domain is a soluble TGF- β receptor.

Embodiment G128 the multispecific chimeric polypeptide of embodiment G127, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G129 the multi-chain chimeric polypeptide of embodiment G128, wherein the second target binding domain comprises a soluble CD137L protein.

Embodiment G130 the multi-chain chimeric polypeptide of embodiment G129, wherein the soluble CD137L protein is soluble human CD137L.

Embodiment G131 the multi-chain chimeric polypeptide of any one of embodiments G126 to G130, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to TGF- β.

Embodiment G132 the multispecific chimeric polypeptide of embodiment G131, wherein the additional target binding domain is a soluble TGF- β receptor.

Embodiment G133 the multispecific chimeric polypeptide of embodiment G132, wherein the soluble TGF- β receptor is a soluble tgfbetarii receptor.

Embodiment G134 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 207.

Embodiment G135 the multi-chain chimeric polypeptide of embodiment G134, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 207.

Embodiment G136 the multi-chain chimeric polypeptide of embodiment G135, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 207.

Embodiment G137 the multi-chain chimeric polypeptide of embodiment G136, wherein the first chimeric polypeptide comprises SEQ ID NO. 207.

Embodiment G138 the multi-chain chimeric polypeptide of embodiment G137, wherein the first chimeric polypeptide comprises SEQ ID NO. 209.

Embodiment G139 the multi-chain chimeric polypeptide of any one of embodiments G1 and G134 to G138, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 232.

Embodiment G140 the multi-chain chimeric polypeptide of embodiment G139, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID No. 232.

Embodiment G141 the multi-chain chimeric polypeptide of embodiment G140, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 232.

Embodiment G142 the multi-chain chimeric polypeptide of embodiment G141, wherein the second chimeric polypeptide comprises SEQ ID NO. 232.

Embodiment G143 the multi-chain chimeric polypeptide of embodiment G142, wherein the second chimeric polypeptide comprises SEQ ID NO. 234.

Embodiment G144 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 236.

Embodiment G145 the multi-chain chimeric polypeptide of embodiment G144, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 236.

Embodiment G146 the multi-chain chimeric polypeptide of embodiment G145, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 236.

Embodiment G147. The multi-chain chimeric polypeptide of embodiment G146, wherein the first chimeric polypeptide comprises SEQ ID NO. 236.

Embodiment G148 the multi-chain chimeric polypeptide of embodiment G147, wherein the first chimeric polypeptide comprises SEQ ID NO. 238.

Embodiment G149 the multi-chain chimeric polypeptide of any one of embodiments G1 and G144 to G148, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 232.

Embodiment G150 the multi-chain chimeric polypeptide of embodiment G149, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 232.

Embodiment G151 the multi-chain chimeric polypeptide of embodiment G150, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 232.

Embodiment G152 the multi-chain chimeric polypeptide of embodiment G151, wherein the second chimeric polypeptide comprises SEQ ID NO. 232.

Embodiment G153 the multi-chain chimeric polypeptide of embodiment G152, wherein the second chimeric polypeptide comprises SEQ ID NO. 234.

Embodiment G154 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 207.

Embodiment G155 the multi-chain chimeric polypeptide of embodiment G154, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 207.

Embodiment G156 the multi-chain chimeric polypeptide of embodiment G155, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 207.

Embodiment G157 the multi-chain chimeric polypeptide of embodiment G156, wherein the first chimeric polypeptide comprises SEQ ID NO. 207.

Embodiment G158 the multi-chain chimeric polypeptide of embodiment G157, wherein the first chimeric polypeptide comprises SEQ ID NO. 209.

Embodiment G159 the multi-chain chimeric polypeptide of any one of embodiments G1 and G154 to G158, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 203.

Embodiment G160. The multi-chain chimeric polypeptide of embodiment G159, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 203.

Embodiment G161 the multi-chain chimeric polypeptide of embodiment G160, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 203.

Embodiment G162. The multi-chain chimeric polypeptide of embodiment G161, wherein the second chimeric polypeptide comprises SEQ ID NO. 203.

Embodiment G163 the multi-chain chimeric polypeptide of embodiment G162, wherein the second chimeric polypeptide comprises SEQ ID NO. 250.

Embodiment G164. The multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 236.

Embodiment G165 the multi-chain chimeric polypeptide of embodiment G164, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 236.

Embodiment G166. The multi-chain chimeric polypeptide of embodiment G165, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID No. 236.

Embodiment G167 the multi-chain chimeric polypeptide of embodiment G166, wherein the first chimeric polypeptide comprises SEQ ID NO. 236.

Embodiment G168 the multi-chain chimeric polypeptide of embodiment G167, wherein the first chimeric polypeptide comprises SEQ ID NO. 238.

Embodiment G169 the multi-chain chimeric polypeptide of any one of embodiments G1 and G164 to G168, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 193.

Embodiment G170. The multi-chain chimeric polypeptide of embodiment G169, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 193.

Embodiment G171 the multi-chain chimeric polypeptide of embodiment G170, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 193.

Embodiment G172 the multi-chain chimeric polypeptide of embodiment G171, wherein the second chimeric polypeptide comprises SEQ ID NO. 193.

Embodiment G173 the multi-chain chimeric polypeptide of embodiment G172, wherein the second chimeric polypeptide comprises SEQ ID NO. 195.

Embodiment G174 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 207.

Embodiment G175. The multi-chain chimeric polypeptide of embodiment G174, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 207.

Embodiment G176 the multi-chain chimeric polypeptide of embodiment G175, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 207.

Embodiment G177 the multi-chain chimeric polypeptide of embodiment G176, wherein the first chimeric polypeptide comprises SEQ ID NO. 207.

Embodiment G178 the multi-chain chimeric polypeptide of embodiment G177, wherein the first chimeric polypeptide comprises SEQ ID NO. 209.

Embodiment G179 the multi-chain chimeric polypeptide of any one of embodiments G1 and G174 to G178, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 268.

Embodiment G180. The multi-chain chimeric polypeptide of embodiment G179, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 268.

Embodiment G181 the multi-chain chimeric polypeptide of embodiment G180, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 268.

Embodiment G182 the multi-chain chimeric polypeptide of embodiment G181, wherein the second chimeric polypeptide comprises SEQ ID NO. 268.

Embodiment G183 the multi-chain chimeric polypeptide of embodiment G182, wherein the second chimeric polypeptide comprises SEQ ID NO. 270.

Embodiment G184. The multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 207.

Embodiment G185 the multi-chain chimeric polypeptide of embodiment G184, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 207.

Embodiment G186 the multi-chain chimeric polypeptide of embodiment G185, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID No. 207.

Embodiment G187 the multi-chain chimeric polypeptide of embodiment G186, wherein the first chimeric polypeptide comprises SEQ ID NO. 207.

Embodiment G188. The multi-chain chimeric polypeptide of embodiment G187, wherein the first chimeric polypeptide comprises SEQ ID NO. 209.

Embodiment G189 the multi-chain chimeric polypeptide of any one of embodiments G1 and G184 to G188, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 272.

Embodiment G190 the multi-chain chimeric polypeptide of embodiment G189, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO 272.

Embodiment G191 the multi-chain chimeric polypeptide of embodiment G190, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO 272.

Embodiment G192. The multi-chain chimeric polypeptide of embodiment G191, wherein the second chimeric polypeptide comprises SEQ ID NO:272.

Embodiment G193 the multi-chain chimeric polypeptide of embodiment G192 wherein the second chimeric polypeptide comprises SEQ ID NO 272.

Embodiment G194 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 207.

Embodiment G195. The multi-chain chimeric polypeptide of embodiment G194, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 207.

Embodiment G196. The multi-chain chimeric polypeptide of embodiment G195, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 207.

Embodiment G197 the multi-chain chimeric polypeptide of embodiment G196, wherein the first chimeric polypeptide comprises SEQ ID NO. 207.

Embodiment G198 the multi-chain chimeric polypeptide of embodiment G197, wherein the first chimeric polypeptide comprises SEQ ID NO. 209.

Embodiment G199 the multi-chain chimeric polypeptide of any one of embodiments G1 and G194 to G198, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 193.

Embodiment G200 the multi-chain chimeric polypeptide of embodiment G199, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 193.

Embodiment G201 the multi-chain chimeric polypeptide of embodiment G200, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 193.

Embodiment G202 the multi-chain chimeric polypeptide of embodiment G201, wherein the second chimeric polypeptide comprises SEQ ID NO. 193.

Embodiment G203 the multi-chain chimeric polypeptide of embodiment G202, wherein the second chimeric polypeptide comprises SEQ ID NO. 195.

Embodiment G204 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 236.

Embodiment G205 the multi-chain chimeric polypeptide of embodiment G204, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 236.

Embodiment G206 the multi-chain chimeric polypeptide of embodiment G205, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 236.

Embodiment G207 the multi-chain chimeric polypeptide of embodiment G206, wherein the first chimeric polypeptide comprises SEQ ID NO. 236.

Embodiment G208 the multi-chain chimeric polypeptide of embodiment G207, wherein the first chimeric polypeptide comprises SEQ ID NO. 238.

Embodiment G209 the multi-chain chimeric polypeptide of any one of embodiments G1 and G204 to G208, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 268.

Embodiment G210 the multi-chain chimeric polypeptide of embodiment G209, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 268.

Embodiment G211 the multi-chain chimeric polypeptide of embodiment G210, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 268.

Embodiment G212 the multi-chain chimeric polypeptide of embodiment G211, wherein the second chimeric polypeptide comprises SEQ ID NO. 268.

Embodiment G213 the multi-chain chimeric polypeptide of embodiment G212, wherein the second chimeric polypeptide comprises SEQ ID NO. 270.

Embodiment G214 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 236.

Embodiment G215 the multi-chain chimeric polypeptide of embodiment G214, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 236.

Embodiment G216 the multi-chain chimeric polypeptide of embodiment G215, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 236.

Embodiment G217 the multi-chain chimeric polypeptide of embodiment G216, wherein the first chimeric polypeptide comprises SEQ ID NO. 236.

Embodiment G218 the multi-chain chimeric polypeptide of embodiment G217, wherein the first chimeric polypeptide comprises SEQ ID NO. 238.

Embodiment G219 the multi-chain chimeric polypeptide of any one of embodiments G1 and G214 to G218, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 300.

Embodiment G220 the multi-chain chimeric polypeptide of embodiment G219 wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 300.

Embodiment G221 the multi-chain chimeric polypeptide of embodiment G220, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 300.

Embodiment G222. The multi-chain chimeric polypeptide of embodiment G221, wherein the second chimeric polypeptide comprises SEQ ID NO. 300.

Embodiment G223 the multi-chain chimeric polypeptide of embodiment G222, wherein the second chimeric polypeptide comprises SEQ ID NO. 302.

Embodiment G224 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 236.

Embodiment G225 the multi-chain chimeric polypeptide of embodiment G224, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 236.

Embodiment G226 the multi-chain chimeric polypeptide of embodiment G225, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 236.

Embodiment G227 the multi-chain chimeric polypeptide of embodiment G226, wherein the first chimeric polypeptide comprises SEQ ID NO. 236.

Embodiment G228. The multi-chain chimeric polypeptide of embodiment G227, wherein the first chimeric polypeptide comprises SEQ ID NO. 238.

Embodiment G229 the multi-chain chimeric polypeptide of any one of embodiments G1 and G224 to G228, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 308.

Embodiment G230 the multi-chain chimeric polypeptide of embodiment G229, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 308.

Embodiment G231 the multi-chain chimeric polypeptide of embodiment G230, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 308.

Embodiment G232 the multi-chain chimeric polypeptide of embodiment G231 wherein the second chimeric polypeptide comprises SEQ ID NO. 308.

Embodiment G233 the multi-chain chimeric polypeptide of embodiment G232, wherein the second chimeric polypeptide comprises SEQ ID NO. 310.

Embodiment G234 the multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 236.

Embodiment G235 the multi-chain chimeric polypeptide of embodiment G234, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 236.

Embodiment G236 the multi-chain chimeric polypeptide of embodiment G235, wherein the first chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 236.

Embodiment G237 the multi-chain chimeric polypeptide of embodiment G236, wherein the first chimeric polypeptide comprises SEQ ID NO. 236.

Embodiment G238 the multi-chain chimeric polypeptide of embodiment G237, wherein the first chimeric polypeptide comprises SEQ ID NO. 238.

Embodiment G239 the multi-chain chimeric polypeptide of any one of embodiments G1 and G234 to G238, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 316.

Embodiment G240. The multi-chain chimeric polypeptide of embodiment G239, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID No. 316.

Embodiment G241 the multi-chain chimeric polypeptide of embodiment G240, wherein the second chimeric polypeptide comprises a sequence at least 95% identical to SEQ ID NO. 316.

Embodiment G242 the multi-chain chimeric polypeptide of embodiment G241 wherein the second chimeric polypeptide comprises SEQ ID NO. 316.

Embodiment G243. The multi-chain chimeric polypeptide of embodiment G242, wherein the second chimeric polypeptide comprises SEQ ID NO. 318.

Embodiment G244 the multi-chain chimeric polypeptide of any one of embodiments G1 to G133, wherein the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more additional antigen binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment G245. The multi-chain chimeric polypeptide of embodiment G244, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more other antigen binding domains, and/or a linker sequence between at least one of the one or more other antigen binding domains and the first domain in the pair of affinity domains.

Embodiment G246 the multi-chain chimeric polypeptide of any one of embodiments G1 to G133, wherein the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and/or C-terminus of the first chimeric polypeptide.

Embodiment G247 the multi-chain chimeric polypeptide of embodiment G246, wherein in the first chimeric polypeptide at least one of the one or more additional target binding domains directly abuts the first domain in the pair of affinity domains.

Embodiment G248 the multi-chain chimeric polypeptide of embodiment G246, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains.

Embodiment G249. The multi-chain chimeric polypeptide of embodiment G246, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains directly adjoins the first target binding domain.

Embodiment G250 the multi-chain chimeric polypeptide of embodiment G246, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain.

Embodiment G251. The multi-chain chimeric polypeptide of embodiment G246, wherein at least one of the one or more additional target binding domains is disposed N-terminal and/or C-terminal to the first chimeric polypeptide, and at least one of the one or more additional target binding domains is located between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment G252. The multi-chain chimeric polypeptide of embodiment G251, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed N-terminally directly abuts the first target binding domain or the first domain of the pair of affinity domains.

Embodiment G253 the multi-chain chimeric polypeptide of embodiment G251, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment G254 the multi-chain chimeric polypeptide of embodiment G251, wherein in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed at the C-terminus directly abuts the first target binding domain or the first domain in the pair of affinity domains.

Embodiment G255 the multi-chain chimeric polypeptide of embodiment G251, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first domain in the first target binding domain or the pair of affinity domains.

Embodiment G256. The multi-chain chimeric polypeptide of embodiment G251, wherein at least one of the one or more additional target binding domains located between the soluble tissue factor domain and the first domain of the pair of affinity domains directly adjoins the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment G257. The multi-chain chimeric polypeptide of embodiment G251, wherein the first chimeric polypeptide further comprises a linker sequence disposed as follows: (i) Between the soluble tissue factor domain and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains is located between the soluble tissue factor domain and the first domain of the pair of affinity domains; and/or (ii) between a first domain of the pair of affinity domains and at least one of the one or more additional target binding domains, at least one of the one or more additional target binding domains being located between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment G258 the multi-chain chimeric polypeptide of any one of embodiments G44 to G46, G57 to G59, G76 to G78, G96 to G98, G109 to G111, G122 to G124, and G131 to G133, wherein the second chimeric polypeptide further comprises an additional target binding domain at the N-terminus or C-terminus of the second chimeric polypeptide.

Embodiment G259 the multi-chain chimeric polypeptide of embodiment G258, wherein in the second chimeric polypeptide the additional target binding domain directly abuts the second domain in the pair of affinity domains.

Embodiment G260. The multi-chain chimeric polypeptide of embodiment G258, wherein the second chimeric polypeptide further comprises a linker sequence between the additional target binding domain in the second chimeric polypeptide and the second domain in the pair of affinity domains.

Embodiment G261 the multi-chain chimeric polypeptide of embodiment G258, wherein in the second chimeric polypeptide the additional target binding domain directly abuts the second target binding domain.

Embodiment G262. The multi-chain chimeric polypeptide of embodiment G258, wherein the second chimeric polypeptide further comprises a linker sequence between the additional target binding domain in the second chimeric polypeptide and the second target binding domain.

Embodiment G263 a composition comprising any of the multiple chain chimeric polypeptides of embodiments G1 to G262.

Embodiment G264. the composition of embodiment G263, wherein the composition is a pharmaceutical composition.

Embodiment G265 a kit comprising at least one dose of the composition of embodiment G263 or G264.

Embodiment G266 a nucleic acid encoding any one of the multi-chain chimeric polypeptides of any one of embodiments G1 to G262.

Embodiment G267 a vector comprising the nucleic acid of embodiment G266.

Embodiment G268 the vector of embodiment G267, wherein the vector is an expression vector.

Embodiment G269 a cell comprising a nucleic acid of embodiment G323 or a vector of embodiment G267 or G268.

Embodiment g270. A method of producing a multi-chain chimeric polypeptide, the method comprising:

culturing the cells of embodiment G269 in a culture medium under conditions sufficient to cause production of the multi-chain chimeric polypeptide; and

recovering the multi-chain chimeric polypeptide from the cells and/or culture medium.

Embodiment G271 a multi-chain chimeric polypeptide produced by the method of embodiment G270.

Embodiment G272 the multi-chain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 97.

Embodiment G273. The multi-chain chimeric polypeptide of embodiment G272, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 97.

Embodiment G274. The multi-chain chimeric polypeptide of embodiment G273, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 97.

Embodiment G275 the multi-chain chimeric polypeptide of embodiment G274, wherein the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO 97.

Embodiment G276 the multi-chain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 98.

Embodiment G277. The multi-chain chimeric polypeptide of embodiment G276, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 98.

Embodiment G278. The multi-chain chimeric polypeptide of embodiment G277, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID No. 98.

Embodiment G279 the multi-chain chimeric polypeptide of embodiment G278 wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO. 98.

Embodiment h1 a method of treating an aging-related disease or disorder in a subject in need thereof, the method comprising administering to a subject identified as having an aging-related disease or disorder a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Embodiment h2 a method of killing or reducing the number of senescent cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more NK cell activators.

Embodiment H3. the method of embodiment H2, wherein the senescent cell is a senescent cancer cell, a senescent monocyte, a senescent lymphocyte, a senescent astrocyte, a senescent microglial cell, a senescent neuron, a senescent tissue fibroblast, a senescent dermal fibroblast, a senescent keratinocyte, or other differentiated tissue-specific dividing functional cell.

Embodiment H4. the method of embodiment H3, wherein the senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced senescent cells.

Embodiment H5. the method of embodiment H2, wherein the subject has been identified or diagnosed with an aging-related disease or disorder.

Embodiment H6. the method of embodiment H1 or H5, wherein the aging-related disease or disorder is selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, skin diseases, premature senility diseases and fragile diseases.

Embodiment H7. the method of embodiment H6, wherein the cancer is selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment H8. the method of embodiment H6, wherein the autoimmune disease is type 1 diabetes.

Embodiment H9. the method of embodiment H6, wherein the metabolic disease is selected from the group consisting of obesity, lipodystrophy, and type 2 diabetes.

Embodiment H10 the method of embodiment H6, wherein the neurodegenerative disease is selected from the group consisting of alzheimer's disease, parkinson's disease, and dementia.

Embodiment H11 the method of embodiment H6, wherein the cardiovascular disease is selected from the group consisting of coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

Embodiment H12 the method of embodiment H6, wherein the skin disorder is selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo, thinning of skin, xeroderma pigmentosum and congenital dysplastic keratosis.

Embodiment H13 the method of embodiment H6, wherein the premature senility disease is selected from the group consisting of premature senility and the premature senility syndrome of caucasian-Ji Erfu.

Embodiment H14 the method of embodiment H6, wherein the fragile disease is selected from the group consisting of fragility, vaccination response, osteoporosis, and sarcopenia.

Embodiment H15. The method of embodiment H1 or H5, wherein the aging-related disease or disorder is selected from the group consisting of: age-related macular degeneration, osteoarthritis, lipoatrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related kidney dysfunction, and chemically induced kidney dysfunction.

Embodiment H16 the method of embodiment H1 or H5, wherein the aging-related disease or disorder is type 2 diabetes or atherosclerosis.

Embodiment H17 the method of any one of embodiments H1 to H16, wherein the administration results in a decrease in the number of senescent cells in the target tissue of the subject.

Embodiment H18 the method of embodiment H17, wherein the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature, bone tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

Embodiment H19 the method of any one of embodiments H1 to H18, wherein the administration results in an increase in the expression level of CD25, CD69, MTOR-C1, SREBP1, IFN- γ, and granzyme B in the activated NK cells.

Embodiment h20 a method of treating an aging-related disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of activated NK cells to a subject identified as having an aging-related disease or disorder.

Embodiment h21 a method of killing or reducing the number of senescent cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of activated NK cells.

Embodiment H2 the method of embodiment H21, wherein the senescent cell is a senescent cancer cell, a senescent monocyte, a senescent lymphocyte, a senescent astrocyte, a senescent microglial cell, a senescent neuron, a senescent tissue fibroblast, a senescent dermal fibroblast, a senescent keratinocyte, or other differentiated tissue-specific dividing functional cell.

Embodiment H3 the method of embodiment H22, wherein the senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced senescent cells.

Embodiment H24 the method of embodiment H21, wherein the subject has been identified or diagnosed with an aging-related disease or disorder.

Embodiment H25. The method of embodiment H20 or H24, wherein the aging-related disease or disorder is selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, skin diseases, premature senility diseases and fragile diseases.

Embodiment H26 the method of embodiment H25, wherein the cancer is selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphoblastic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric cancer and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer and hepatocellular carcinoma.

Embodiment H7 the method of embodiment H25, wherein the autoimmune disease is type 1 diabetes.

Embodiment H28 the method of embodiment H25, wherein the metabolic disease is selected from the group consisting of obesity, lipodystrophy, and type 2 diabetes.

Embodiment H9 the method of embodiment H25, wherein the neurodegenerative disease is selected from the group consisting of alzheimer's disease, parkinson's disease, and dementia.

Embodiment H30 the method of embodiment H25, wherein the cardiovascular disease is selected from the group consisting of coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

Embodiment H31 the method of embodiment H25, wherein the skin disorder is selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo, thinning of skin, xeroderma pigmentosum and congenital dysplastic keratosis.

Embodiment H32 the method of embodiment H25, wherein the premature senility disease is selected from the group consisting of premature senility and the premature senility syndrome of caucasian-Ji Erfu.

Embodiment H33 the method of embodiment H25, wherein the fragile disease is selected from the group consisting of fragility, vaccination response, osteoporosis, and sarcopenia.

Embodiment H34. The method of embodiment H20 or H24, wherein the aging-related disease or disorder is selected from the group consisting of: age-related macular degeneration, osteoarthritis, lipoatrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related kidney dysfunction, and chemically induced kidney dysfunction.

Embodiment H35. the method of any one of embodiments H20 to H34, wherein the method further comprises:

obtaining resting NK cells; and

contacting the resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein the contacting results in the production of activated NK cells, which are subsequently administered to the subject.

Embodiment H36 the method of embodiment H35, wherein the resting NK cells are autologous NK cells obtained from the subject.

Embodiment H37 the method of embodiment H35, wherein the resting NK cells are allogeneic resting NK cells.

Embodiment H38 the method of embodiment H35, wherein the resting NK cells are artificial NK cells.

Embodiment H39 the method of embodiment H35, wherein the resting NK cells are haplotype resting NK cells.

Embodiment H40 the method of any one of embodiments H35 to H39, wherein the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

Embodiment H41 the method of any one of embodiments H35 to H40, wherein the method further comprises isolating the activated NK cells, followed by administering the activated NK cells to the subject.

Embodiment h42 a method of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Embodiment h43 a method of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of activated NK cells.

Embodiment H44 the method of embodiment H43, wherein the method further comprises:

obtaining resting NK cells; and

contacting the resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein the contacting results in the production of activated NK cells, which are subsequently administered to the subject.

Embodiment H45 the method of embodiment H44, wherein the resting NK cells are autologous NK cells obtained from the subject.

Embodiment H46 the method of embodiment H44, wherein the resting NK cells are allogeneic resting NK cells.

Embodiment H47 the method of embodiment H44, wherein the resting NK cells are artificial NK cells.

Embodiment H48 the method of embodiment H44, wherein the resting NK cells are haplotype resting NK cells.

Embodiment H49 the method of any one of embodiments H44 to H48, wherein the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

Embodiment H50 the method of any one of embodiments H44 to H49, wherein the method further comprises isolating the activated NK cells, followed by administering the activated NK cells to the subject.

Embodiment H51 the method of any one of embodiments H42 to H50, wherein the method results in an improvement in skin texture and/or appearance of the subject over a period of time.

Embodiment H52 the method of embodiment H51, wherein the method reduces the rate of skin wrinkle formation in the subject over a period of time.

Embodiment H53 the method of embodiment H51 or H52, wherein the method results in improved skin coloration of the subject over a period of time.

Embodiment H54 the method of any one of embodiments H51-H53, wherein the method results in an improvement in skin texture of the subject over a period of time.

Embodiment H55 the method of any one of embodiments H42 to H50, wherein the method results in an improvement in hair texture and/or appearance of the subject over a period of time.

Embodiment H56 the method of embodiment H55, wherein the method reduces the rate of white hair formation in the subject over a period of time.

Embodiment H57 the method of embodiment H55 or H56, wherein the method reduces the number of white hairs in the subject over a period of time.

Embodiment H58 the method of any one of embodiments H55 to H57, wherein the method results in a decrease in the rate of hair loss in the subject over time.

Embodiment H59 the method of any one of embodiments H55-H58, wherein the method results in an improvement in hair texture in the subject over a period of time.

Embodiment H60 the method of any one of embodiments H42 to H59, wherein the period of time is between about one month and about year.

Embodiment H61 the method of any one of embodiments H42 to H60, wherein the method reduces the number of aged dermal fibroblasts in the skin of the subject over a period of time.

Embodiment h62 a method of aiding in the treatment of obesity in a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Embodiment h63 a method of contributing to the treatment of obesity in a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of activated NK cells.

Embodiment H64 the method of embodiment H63, wherein the method further comprises:

obtaining resting NK cells; and

contacting the resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein the contacting results in the production of activated NK cells, which are subsequently administered to the subject.

Embodiment H65 the method of embodiment H64, wherein the resting NK cells are autologous NK cells obtained from the subject.

Embodiment H66 the method of embodiment H64, wherein the resting NK cells are allogeneic resting NK cells.

Embodiment H67 the method of embodiment H64, wherein the resting NK cells are artificial NK cells.

Embodiment H68 the method of embodiment H64, wherein the resting NK cells are haplotype resting NK cells.

Embodiment H69 the method of any one of embodiments H64 to H68, wherein the resting NK cells are genetically engineered NK cells bearing a chimeric antigen receptor or a recombinant T cell receptor.

Embodiment H70 the method of any one of embodiments H64 to H69, wherein the method further comprises isolating the activated NK cells, followed by administering the activated NK cells to the subject.

Embodiment H71 the method of any one of embodiments H62-H70, wherein the method causes the mass of the subject to decrease over a period of time.

Embodiment H72 the method of any one of embodiments H62-H71, wherein the method results in a decrease in the Body Mass Index (BMI) of the subject over a period of time.

Embodiment H73 the method of any one of embodiments H62 to H70, wherein the method reduces the rate of progression of pre-diabetes to type 2 diabetes in the subject.

Embodiment H74 the method of any one of embodiments H62 to H70, wherein the method results in a decrease in fasting serum glucose levels in the subject.

Embodiment H75 the method of any one of embodiments H62 to H70, wherein the method increases insulin sensitivity in the subject.

Embodiment H76 the method of any one of embodiments H62 to H70, wherein the method reduces the severity of atherosclerosis in the subject.

Embodiment H77 the method of any one of embodiments H62-H76, wherein the period of time is between about two weeks to about 10 years.

Embodiment H78. The method of any one of embodiments H1 to H19, H35 to H42, H44 to H62, and H64 to H77, wherein at least one of the one or more NK cell activators causes activation of one or more of: IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor, IL-33 receptor; receptors for CD16, CD69, CD25, CD36, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS5 and KIR3DS 1.

Embodiment H79 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the IL-2 receptor is soluble IL-2 or an agonistic antibody that specifically binds to the IL-2 receptor.

Embodiment H80 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the IL-7 receptor is soluble IL-7 or an agonistic antibody that specifically binds to the IL-7 receptor.

Embodiment H81 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the IL-12 receptor is soluble IL-12 or an agonistic antibody that specifically binds to the IL-12 receptor.

Embodiment H82 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the IL-15 receptor is soluble IL-15 or an agonistic antibody that specifically binds to the IL-15 receptor.

Embodiment H83 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the IL-21 receptor is soluble IL-21 or an agonistic antibody that specifically binds to the IL-21 receptor.

Embodiment H84 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the IL-33 receptor is soluble IL-33 or an agonistic antibody that specifically binds to the IL-33 receptor.

Embodiment H85 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the CD16 receptor is an agonistic antibody that specifically binds to CD 16.

Embodiment H86 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of CD69 receptor is an agonistic antibody that specifically binds to CD 69.

Embodiment H87 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the receptor for CD25, CD36, CD59 is an agonistic antibody that specifically binds to CD25, CD6, CD 59.

Embodiment H88 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of CD352 receptor is an agonistic antibody that specifically binds to CD 352.

Embodiment H89 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the NKp80 receptor is an agonistic antibody that specifically binds to NKp 80.

Embodiment H90 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of DNAM-1 receptors is an agonistic antibody that specifically binds to DNAM-1.

Embodiment H91 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the 2B4 receptor is an agonistic antibody that specifically binds to 2B 4.

Embodiment H92 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the NKp30 receptor is an agonistic antibody that specifically binds to NKp 30.

Embodiment H93 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the NKp44 receptor is an agonistic antibody that specifically binds to NKp 44.

Embodiment H94 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the NKp46 receptor is an agonistic antibody that specifically binds to NKp 46.

Embodiment H95 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of the NKG2D receptor is an agonistic antibody that specifically binds to NKG 2D.

Embodiment H96 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of KIR2DS1 receptor is an agonistic antibody that specifically binds to KIR2DS 1.

Embodiment H97 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of KIR2DS2/3 receptors is an agonistic antibody that specifically binds to KIR2DS 2/3.

Embodiment H98 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of KIR2DL4 receptor is an agonistic antibody that specifically binds to KIR2DL 4.

Embodiment H99 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of KIR2DS4 receptor is an agonistic antibody that specifically binds to KIR2DS 4.

Embodiment H100 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of KIR2DS5 receptor is an agonistic antibody that specifically binds to KIR2DS 5.

Embodiment H101 the method of embodiment H78, wherein at least one of the one or more NK cell activators that cause activation of KIR3DS1 receptor is an agonistic antibody that specifically binds to KIR3DS 1.

Embodiment H102. The method of any one of embodiments H1 to H19, H35 to H42, H44 to H62, and H64 to H101, wherein at least one of the one or more NK cell activators causes a decrease in activation of one or more of: PD-1, TGF-beta receptor, TIGIT, CD1, TIM-3, siglec-7, IRP60, tactive, IL1R8, NKG2A/KLRD1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1 and LAG-2.

Embodiment H103 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of PD-1 is an antagonistic antibody that specifically binds to PD-1, soluble PD-L1, or an antibody that specifically binds to PD-L1.

Embodiment H104 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of the TGF- β receptor is a soluble TGF- β receptor, an antibody that specifically binds to TGF- β, or an antagonistic antibody that specifically binds to the TGF- β receptor.

Embodiment H105 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced TIGIT activation is an antagonistic antibody that specifically binds to TIGIT, a soluble TIGIT, or an antibody that specifically binds to TIGIT ligand.

Embodiment H106 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of CD1 is an antagonistic antibody that specifically binds to CD1, soluble CD1, or an antibody that specifically binds to a CD1 ligand.

Embodiment H107 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of TIM-3 is an antagonistic antibody that specifically binds to TIM-3, soluble TIM-3, or an antibody that specifically binds to a TIM-3 ligand.

Embodiment H108 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of Siglec-7 is an antagonistic antibody that specifically binds to Siglec-7, or an antibody that specifically binds to a Siglec-7 ligand.

Embodiment H109 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of IRP60 is an antagonistic antibody that specifically binds to IRP60, or an antibody that specifically binds to IRP60 ligand.

Embodiment H110 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of Tactile is an antagonistic antibody that specifically binds to Tactile, or an antibody that specifically binds to Tactile ligand.

Embodiment H111 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of IL1R8 is an antagonistic antibody that specifically binds to IL1R8, or an antibody that specifically binds to an IL1R8 ligand.

Embodiment H112 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of NKG2A/KLRD1 is an antagonistic antibody that specifically binds to NKG2A/KLRD1, or an antibody that specifically binds to a NKG2A/KLRD1 ligand.

Embodiment H113 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced KIR2DL1 activation is an antagonistic antibody that specifically binds to KIR2DL1, or an antibody that specifically binds to a KIR2DL1 ligand.

Embodiment H114 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of KIR2DL2/3 is an antagonistic antibody that specifically binds to KIR2DL2/3, or an antibody that specifically binds to a KIR2DL2/3 ligand.

Embodiment H115 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced KIR2DL5 activation is an antagonistic antibody that specifically binds to KIR2DL5, or an antibody that specifically binds to a KIR2DL5 ligand.

Embodiment H116 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced KIR3DL1 activation is an antagonistic antibody that specifically binds to KIR3DL1, or an antibody that specifically binds to KIR3DL1 ligand.

Embodiment H117 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced KIR3DL2 activation is an antagonistic antibody that specifically binds to KIR3DL2, or an antibody that specifically binds to KIR3DL2 ligand.

Embodiment H118 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause reduced activation of ILT2/LIR-1 is an antagonistic antibody that specifically binds to ILT2/LIR-1, or an antibody that specifically binds to a ligand of ILT 2/LIR-1.

Embodiment H119 the method of embodiment H102, wherein at least one of the one or more NK cell activators that cause decreased activation of LAG-2 is an antagonistic antibody that specifically binds to LAG-2, or an antibody that specifically binds to LAG-2 ligand.

Embodiment H120 the method of any one of embodiments H1 to H19, H35 to H42, H44 to H62, and H64 to H77, wherein at least one of the one or more NK cell activators is a single chain chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A second target binding domain.

Embodiment H121 the multi-chain chimeric polypeptide of embodiment H120, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other.

Embodiment H122 the method of embodiment H120, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain.

Embodiment H123 the method of any one of embodiments H120 to H122, wherein the soluble tissue factor domain and the second target binding domain are directly adjacent to each other.

Embodiment H124 the method of any one of embodiments H120 to H122, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain.

Embodiment H125 the method of embodiment H120, wherein the first target binding domain and the second target binding domain are directly adjacent to each other.

Embodiment H126 the method of embodiment H120, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain.

Embodiment H127 the method of embodiment H125 or H126, wherein the second target binding domain and the soluble tissue factor domain are directly adjacent to each other.

Embodiment H128 the method of embodiment H125 or H126, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the second target binding domain and the soluble tissue factor domain.

Embodiment H129 the method of any one of embodiments H120 to H128 wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

Embodiment H130 the method of embodiment H129, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment H131 the method of embodiment H130, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment H132 the method of any one of embodiments H120 to H128, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

Embodiment H133 the method of any one of embodiments H120 to H132, wherein one or both of the first target binding domain and the second target binding domain is an antigen binding domain.

Embodiment H134 the method of embodiment H133, wherein the first target binding domain and the second target binding domain are each an antigen binding domain.

Embodiment H135 the method of embodiment H134, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment H136 the method of any one of embodiments H120 to H135, wherein one or both of the first target binding domain and the second target binding domain bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, TGF-beta receptor II (TGF-. Beta.RII) ligand, TGF-. Beta.RIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, PDGF-DD receptor, receptor (SCF-DD receptor), receptor (receptor 3, MICP receptor) receptor of MIC-3, receptor-like receptor (CD-B) 122, receptor-3 of MIC-cell-like receptor (CD-3, receptor-B-receptor-3, receptor-B-receptor-4.

Embodiment H137 the method of any one of embodiments H120 to H128, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein.

Embodiment H138 the method of embodiment H137, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

Embodiment H139 the method of any one of embodiments H120 to H128, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor.

Embodiment H140 the method of embodiment H139, wherein the soluble interleukin or cytokine receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, soluble receptor for TNF alpha, soluble receptor for IL-4, or soluble receptor for IL-10.

Embodiment H141 the method of any one of embodiments H120 to H140, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H142. The method of embodiment H141, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment H143 the method of embodiment H142, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 93.

Embodiment H144 the method of embodiment H143, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment H145 the method of any one of embodiments H141 to H144, wherein the soluble human tissue factor domain does not comprise one or more of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

Phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment H146 the method of embodiment H145, wherein the soluble human tissue factor domain does not comprise any of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment H147 the method of any one of embodiments H120 to H146, wherein the soluble tissue factor domain is incapable of binding factor vila.

Embodiment H148 the method of any one of embodiments H120 to H147, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment H149 the method of any one of embodiments H120 to H148, wherein the single-chain chimeric polypeptide does not stimulate blood clotting in the mammal.

Embodiment H150 the method of any one of embodiments H120 to H149, wherein the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and/or C-terminus.

Embodiment H151 the method of embodiment H150, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus.

Embodiment H152 the method of embodiment H151, wherein the one or more additional target binding domains directly adjoin the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment H153 the method of embodiment H152, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment H154 the method of embodiment H150, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its C-terminus.

Embodiment H155 the method of embodiment H154, wherein one of the one or more additional target binding domains directly adjoins the first target binding domain, the second target binding domain or the soluble tissue factor domain.

Embodiment H156 the single-chain chimeric polypeptide of embodiment H154, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment H157 the method of embodiment H150, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-and C-termini.

Embodiment H158 the method of embodiment H157, wherein one of the one or more additional antigen binding domains located at the N-terminus directly abuts the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment H159 the method of embodiment H157, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains at the N-terminus and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment H160 the method of embodiment H157, wherein one of the one or more additional antigen binding domains located at the C-terminus directly abuts the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment H161. The method of embodiment H157, wherein the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains at the C-terminus and the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment H162 the method of any one of embodiments H150 to H161, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen.

Embodiment H163 the method of embodiment H162, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope.

Embodiment H164 the method of embodiment H163, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

Embodiment H165 the method of embodiment H162, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen.

Embodiment H166 the method of embodiment H165, wherein the first target binding domain, the second target binding domain and the one or more additional target binding domains each specifically bind to the same epitope.

Embodiment H167 the method of embodiment H166, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

Embodiment H168 the method of any one of embodiments H150 to H161, wherein the first target binding domain, the second target binding domain and the one or more additional target binding domains specifically bind to different antigens.

Embodiment H169 the method of any one of embodiments H150 to H168, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains is an antigen binding domain.

Embodiment H170 the method of embodiment H169, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains.

Embodiment H171 the method of embodiment H170, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment H172. The method of any one of embodiments H150 to H171, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands for TGF-beta receptor II (TGF-beta RII) a ligand for TGF-beta RIII, a ligand for DNAM1, a ligand for NKp46, a ligand for NKp44, a ligand for NKG2D, a ligand for NKp30, a ligand for scMHCI, a ligand for scTCR, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, and a receptor for CD 122.

Embodiment H173 the method of any one of embodiments H150 to H161, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment H174 the method of embodiment H173, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

Embodiment H175 the method of any one of embodiments H150 to H161, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment H176 the method of embodiment H175, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment H177 the method of any one of embodiments H1 to H19, H35 to H42, H44 to H62, and H64 to H77, wherein at least one of the one or more NK cell activators is a multi-chain chimeric polypeptide comprising:

(c) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(d) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of a first domain and a second domain of a pair of affinity domains.

Embodiment H178 the method of embodiment H177, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

Embodiment H179 the method of embodiment H177, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment H180 the method of any one of embodiments H177 to H179, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

Embodiment H181 the method of any one of embodiments H177 to H179, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment H182 the method of any one of embodiments H177 to H181, wherein the second domain of the pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

The method of any one of embodiments H177-H181, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

Embodiment H184 the method of any one of embodiments H177 to H183, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

Embodiment H185 the method of embodiment H184, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment H186 the method of embodiment H185 wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment H187 the method of any one of embodiments H177 to H183, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

Embodiment H188 the method of any one of embodiments H177 to H187, wherein one or both of the first target binding domain and the second target binding domain is an antigen binding domain.

Embodiment H189 the method of embodiment H188, wherein the first target binding domain and the second target binding domain are each an antigen binding domain.

Embodiment H190 the method of embodiment H188 or H189, wherein the antigen binding domain comprises an scFv or a single domain antibody.

Embodiment H191 the method of any of embodiments H177 to H190, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, TGF-beta receptor II (TGF-. Beta.RII) ligand, TGF-. Beta.RIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, PDGF-DD receptor, receptor (SCF-DD receptor), receptor (receptor 3, MICP receptor) receptor of MIC-3, receptor-like receptor (CD-B) 122, receptor-3 of MIC-cell-like receptor (CD-3, receptor-B-receptor-3, receptor-B-receptor-4.

Embodiment H192 the method of any one of embodiments H177 to H183, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein.

Embodiment H193 the method of embodiment H192, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

Embodiment H194 the method of any one of embodiments H177-H183, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor.

Embodiment H195 the method of embodiment H194, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

The method of any one of embodiments H177-H195, wherein the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more additional antigen binding domains is positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

The method of embodiment H197 the method of embodiment H196, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more other antigen binding domains, and/or a linker sequence between at least one of the one or more other antigen binding domains and the first domain of the pair of affinity domains.

Embodiment H198 the method of any one of embodiments H177 to H195, wherein the first chimeric polypeptide further comprises one or more additional target binding domains located at the N-terminus and/or C-terminus of the first chimeric polypeptide.

Embodiment H199. The multi-chain chimeric polypeptide of embodiment H198, wherein at least one of the one or more additional target binding domains directly abuts the first domain in the pair of affinity domains in the first chimeric polypeptide.

Embodiment H200 the method of embodiment H198, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain in the pair of affinity domains.

Embodiment H01 the method of embodiment H198, wherein at least one of the one or more additional target binding domains directly adjoins the first target binding domain in the first chimeric polypeptide.

Embodiment H02 the method of embodiment H198, wherein the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain.

Embodiment H03 the method of embodiment H198, wherein at least one of the one or more additional target binding domains is disposed N-terminal and/or C-terminal to the first chimeric polypeptide and at least one of the one or more additional target binding domains is positioned between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment H04 the method of embodiment H203, wherein at least one additional target binding domain of the one or more additional target binding domains disposed at the N-terminus directly adjoins the first target binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H05 the method of embodiment H203, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains.

Embodiment H06 the method of embodiment H203, wherein at least one of the one or more additional target binding domains disposed at the C-terminus directly abuts the first target binding domain or the first domain of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H07 the method of embodiment H203, wherein the first chimeric polypeptide further comprises a linker sequence disposed between at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains.

Embodiment H08 the method of embodiment H203, wherein at least one of the one or more additional target binding domains located between the soluble tissue factor domain and the first domain of the pair of affinity domains directly adjoins the soluble tissue factor domain and/or the first domain of the pair of affinity domains.

Embodiment H09 the method of embodiment H203, wherein the first chimeric polypeptide further comprises a linker sequence disposed between (i) the soluble tissue factor domain and at least one of the one or more additional target binding domains, the target binding domain being positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains; and/or (ii) between a first domain of a pair of affinity domains and at least one of the one or more additional target binding domains, the target binding domain being positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

Embodiment H10. the method of any one of embodiments H177 to H209, wherein the second chimeric polypeptide further comprises one or more additional target binding domains located at the N-terminus and/or C-terminus of the second chimeric polypeptide.

Embodiment H11 the method of embodiment H210, wherein at least one of the one or more additional target binding domains directly adjoins the second domain of the pair of affinity domains in the second chimeric polypeptide.

Embodiment H12 the method of embodiment H210, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain in the pair of affinity domains.

Embodiment H13 the method of embodiment H210 wherein at least one of the one or more additional target binding domains directly adjoins the second target binding domain in the second chimeric polypeptide.

Embodiment H14 the method of embodiment H210, wherein the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

Embodiment H15 the method of any one of embodiments H196 to H214, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen.

Embodiment H16 the method of embodiment H215, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope.

Embodiment H17 the method of embodiment H216, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

Embodiment H18 the method of embodiment H215, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen.

Embodiment H19 the method of embodiment H218, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope.

Embodiment H20 the method of embodiment H219, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

Embodiment H21 the method of any one of embodiments H196 to H214, wherein the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to different antigens.

Embodiment H22 the method of any one of embodiments H196 to H221, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains.

Embodiment H23 the method of embodiment H222, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains.

Embodiment H24 the method of embodiment H223, wherein the antigen binding domain comprises an scFv.

Embodiment H25 the method of any one of embodiments H196 to H224, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, ligands for TGF-beta receptor II (TGF-beta RII) a ligand for TGF-beta RIII, a ligand for DNAM1, a ligand for NKp46, a ligand for NKp44, a ligand for NKG2D, a ligand for NKp30, a ligand for scMHCI, a ligand for scTCR, a receptor for PDGF-DD, a receptor for Stem Cell Factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT 3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, and a receptor for CD 122.

Embodiment H26 the method of any one of embodiments H196-H214, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment H27 the method of embodiment H226, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

Embodiment H28 the method of any one of embodiments H196-H214, wherein one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment H29 the method of embodiment H228, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment H30 the method of any one of embodiments H196 to H229, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H31 the method of embodiment H230, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

Embodiment H32 the method of embodiment H231, wherein the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO. 93.

Embodiment H33 the method of embodiment H232, wherein the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO. 93.

Embodiment H34 the method of any one of embodiments H230 to H233, wherein the soluble human tissue factor domain does not comprise one or more of:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

Arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment H35 the method of embodiment H234, wherein the soluble human tissue factor domain does not comprise any of the following:

lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;

isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;

tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;

aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;

tyrosine at an amino acid position corresponding to amino acid position 94 of mature wild-type human tissue factor protein;

arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and

phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

Embodiment H36 the method of any one of embodiments H196 to H235, wherein the soluble tissue factor domain is unable to bind to factor vila.

Embodiment H37 the method of any one of embodiments H196-H236, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa.

Embodiment H38 the method of any one of embodiments H196 to H237, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment H39 the method of any one of embodiments H196 to H238, wherein the pair of affinity domains is a sushi domain from the human IL-15 receptor alpha chain (IL-15 Ralpha) and soluble IL-15.

Embodiment H40 the method of embodiment H239, wherein the soluble IL15 has a D8N or D8A amino acid substitution.

Embodiment H41 the method of embodiment H239 or H240, wherein the human IL-15Rα is mature full length IL-15Rα.

Embodiment H42 the method of any one of embodiments H196 to H238, wherein the pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, and SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

Embodiment H43 the method of any one of embodiments H1 to H19, H35 to H42, H44 to H62, and H64 to H77, wherein at least one of the one or more NK cell activators is a multi-chain chimeric polypeptide comprising:

(a) First and second chimeric polypeptides each comprising:

(i) A first target binding domain;

(ii) An Fc domain; and

(iii) A first domain of a pair of affinity domains;

(b) Third and fourth chimeric polypeptides, each comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein the first and second chimeric polypeptides are associated with the third and fourth chimeric polypeptides by binding of a first domain to a second domain in a pair of affinity domains, and the first and second chimeric polypeptides are associated by their Fc domains.

Embodiment H44 the method of embodiment H243, wherein the first target binding domain and the Fc domain are directly adjacent to each other in the first and second chimeric polypeptides.

Embodiment H45 the method of embodiment H243, wherein the first and second chimeric polypeptides further comprise a linker sequence between the first target binding domain and the Fc domain in the first and second chimeric polypeptides.

Embodiment h046 the method of any one of embodiments H243 to H245, wherein the Fc domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first and second chimeric polypeptides.

Embodiment H47 the method of any one of embodiments H243 to H245, wherein the first chimeric polypeptide further comprises a linker sequence between the Fc domain in the first and second chimeric polypeptides and the first domain in the pair of affinity domains.

Embodiment h248 the method of any one of embodiments H243 to H247, wherein the second domain of the pair of affinity domains and the second target binding domain are directly adjacent to each other in the third and fourth chimeric polypeptides.

Embodiment h049 the method of any one of embodiments H243 to H247, wherein the third and fourth chimeric polypeptides further comprise a linker sequence between the second domain in the pair of affinity domains in the third and fourth chimeric polypeptides and the second target binding domain.

Embodiment h250 the method of any one of embodiments H243 to H249, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

Embodiment H251 the method of embodiment H250, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

Embodiment H252 the method of embodiment H251, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

Embodiment H53 the method of any one of embodiments H243 to H249, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

Embodiment H54 the method of any one of embodiments H243 to H253, wherein one or both of the first target binding domain and the second target binding domain is an antigen binding domain.

Embodiment H55 the method of embodiment H254, wherein the first target binding domain and the second target binding domain are each an antigen binding domain.

Embodiment H56 the method of embodiment H254 or H255, wherein the antigen binding domain comprises an scFv or single domain antibody.

Embodiment H57 the method of any one of embodiments H243 to H256, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, TGF-beta receptor II (TGF-. Beta.RII) ligand, TGF-. Beta.RIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, PDGF-DD receptor, receptor (SCF-DD receptor), receptor (receptor 3, MICP receptor) receptor of MIC-3, receptor-like receptor (CD-B) 122, receptor-3 of MIC-cell-like receptor (CD-3, receptor-B-receptor-3, receptor-B-receptor-4.

Embodiment H58 the method of any one of embodiments H243 to H256, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein.

Embodiment H59 the method of embodiment H258, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

Embodiment H60 the method of any one of embodiments H243 to H256, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor.

Embodiment H61 the method of embodiment H260, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment I1. a method of treating an aging-related disease or disorder in a subject in need thereof, the method comprising administering to a subject identified as having an aging-related disease or disorder a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Embodiment i2. A method of killing or reducing the number of senescent cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more NK cell activators.

Embodiment I3 the method of any one of embodiments I1 to I2, wherein the administration results in a decrease in the number of senescent cells in the target tissue of the subject.

Embodiment I4. the method of embodiment I3, wherein the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature, bone tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

Embodiment I5. a method of treating an aging-related disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of activated NK cells to a subject identified as having an aging-related disease or disorder.

Embodiment I6. a method of killing or reducing the number of senescent cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of activated NK cells.

Embodiment I7. the method of any one of embodiments I1 to I6, wherein the subject has been identified or diagnosed with an aging-related disease or disorder.

Embodiment I8. the method of embodiment I7, wherein the aging-related disease or disorder is selected from the group consisting of: cancer, autoimmune diseases, metabolic diseases, neurodegenerative diseases, cardiovascular diseases, skin diseases, premature senility diseases and fragile diseases.

Embodiment I9. the method of embodiment I8, wherein the cancer is selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

Embodiment I10 the method of embodiment I8, wherein the autoimmune disease is type 1 diabetes.

Embodiment I11 the method of embodiment I8 wherein the metabolic disease is selected from the group consisting of obesity, lipodystrophy, and type 2 diabetes.

Embodiment I12 the method of embodiment I8 wherein the neurodegenerative disease is selected from the group consisting of alzheimer's disease, parkinson's disease, and dementia.

Embodiment i13 the method of embodiment 8, wherein the cardiovascular disease is selected from the group consisting of coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

Embodiment I14 the method of embodiment I8, wherein the skin disorder is selected from the group consisting of: wound healing, alopecia, wrinkles, senile lentigo, thinning of skin, xeroderma pigmentosum and congenital dysplastic keratosis.

Embodiment I15 the method of embodiment I8 wherein the premature senility disease is selected from the group consisting of premature senility and a premature respiratory syndrome of michelson-Ji Erfu.

Embodiment I16 the method of embodiment I8 wherein the fragile disease is selected from the group consisting of fragility, vaccination response, osteoporosis, and sarcopenia.

Embodiment I17 the method of any one of embodiments I1 to I6, wherein the aging-related disease or disorder is selected from the group consisting of: osteoarthritis, lipoatrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related kidney dysfunction, and chemically induced kidney dysfunction.

Embodiment I18 the method of any one of embodiments I1 to I6, wherein the aging-related disease or disorder is type 2 diabetes or atherosclerosis.

Embodiment i19 a method of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Embodiment i20 a method of improving the texture and/or appearance of skin and/or hair of a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of activated NK cells.

Embodiment I21 the method of any one of embodiments I19 to I20, wherein the method results in an improvement in the skin texture and/or appearance of the subject over a period of time.

Embodiment I22 the method of embodiment I21, wherein the method reduces the rate of skin wrinkle formation in the subject over a period of time.

Embodiment I23 the method of embodiment I21 or I22, wherein the method results in an improvement in skin coloration of the subject over a period of time.

Embodiment I24 the method of any one of embodiments I21 to I23, wherein the method results in an improvement in skin texture of the subject over a period of time.

Embodiment I25 the method of any one of embodiments I20 to I24, wherein the method results in an improvement in hair texture and/or appearance of the subject over a period of time.

Embodiment I26 the method of embodiment I25, wherein the method reduces the rate of white hair formation in the subject over a period of time.

Embodiment I27 the method of embodiment I25 or I26, wherein the method reduces the number of white hairs in the subject over a period of time.

Embodiment I28 the method of any one of embodiments I25 to I27, wherein the method results in a decrease in the rate of hair loss in the subject over time.

Embodiment I29 the method of any one of embodiments I25 to I28, wherein the method results in an improvement in hair texture in the subject over a period of time.

Embodiment I30 the method of any one of embodiments I19 to I29, wherein the method reduces the number of aged dermal fibroblasts in the skin of the subject over a period of time.

Embodiment i31 a method of aiding in the treatment of obesity in a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of one or more Natural Killer (NK) cell activators.

Embodiment i32 a method of contributing to the treatment of obesity in a subject in need thereof over a period of time, the method comprising administering to the subject a therapeutically effective amount of activated NK cells.

Embodiment I33 the method of any one of embodiments I1 to I32, wherein the method further comprises:

obtaining resting NK cells; and

contacting the resting NK cells in vitro in a liquid medium comprising one or more NK cell activators, wherein the contacting results in the production of activated NK cells, followed by administration to a subject.

Embodiment I34 the method of embodiment I33, wherein the resting NK cells are genetically engineered NK cells carrying a chimeric antigen receptor or a recombinant T cell receptor.

Embodiment i35 the method of embodiment 33, wherein the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the resting NK cells or activated NK cells, followed by administration to the subject.

Embodiment I36 the method of any one of embodiments I31 to I35, wherein the method causes the mass of the subject to decrease over a period of time.

Embodiment I37 the method of any one of embodiments I31-I36, wherein the method results in a decrease in the Body Mass Index (BMI) of the subject over a period of time.

Embodiment I38 the method of any one of embodiments I31 to I35, wherein the method reduces the rate of progression of pre-diabetes to type 2 diabetes in the subject.

Embodiment I39 the method of any one of embodiments I31 to I35, wherein the method results in a decrease in fasting serum glucose levels in the subject.

Embodiment I40 the method of any one of embodiments I31 to I35, wherein the method increases insulin sensitivity in the subject.

Embodiment I41 the method of any one of embodiments I31 to I35, wherein the method results in a reduction in the severity of atherosclerosis in the subject.

Embodiment I42 the method of any one of embodiments I1 to I41, wherein at least one of the one or more NK cell activators causes activation of one or more of: IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor, IL-33 receptor; receptors for CD16, CD69, CD25, CD36, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS5 and KIR3DS 1.

Embodiment I43 the method of any one of embodiments I1 to I42, wherein at least one of the one or more NK cell activators causes a reduction in activation of one or more of: PD-1, TGF-beta receptor, TIGIT, CD1, TIM-3, siglec-7, IRP60, tactive, IL1R8, NKG2A/KLRD1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1 and LAG-2.

Embodiment I44 the method of any one of embodiments I1 to I41, wherein at least one of the one or more NK cell activators is a single chain chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(ii) A second target binding domain.

Embodiment I45 the multi-chain chimeric polypeptide of embodiment I44, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other.

Embodiment I46 the method of embodiment I44, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain.

Embodiment I47 the method of any one of embodiments I44 to I46, wherein the soluble tissue factor domain and the second target binding domain are directly adjacent to each other.

Embodiment I48 the method of any one of embodiments I44 to I46, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain.

Embodiment I49 the method of any one of embodiments I1 to I41, wherein at least one of the one or more NK cell activators is a multi-chain chimeric polypeptide comprising:

(a) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(b) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of a first domain and a second domain of a pair of affinity domains.

Embodiment I50 the method of embodiment I49, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

Embodiment I51 the method of embodiment I49, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

Embodiment I52 the method of any one of embodiments I49 to I51, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

The method of any one of embodiments I53, I49 to I51, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

Embodiment I54 the method of any one of embodiments I49 to I53, wherein the second domain in the pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

Embodiment I55 the method of any one of embodiments I49 to I53, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

Embodiment I56 the method of any one of embodiments I1 to I41, wherein at least one of the one or more NK cell activators is a multi-chain chimeric polypeptide comprising:

(a) First and second chimeric polypeptides each comprising:

(i) A first target binding domain;

(ii) An Fc domain; and

(iii) A first domain of a pair of affinity domains;

(b) Third and fourth chimeric polypeptides, each comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein the first and second chimeric polypeptides are associated with the third and fourth chimeric polypeptides by binding of a first domain to a second domain in a pair of affinity domains, and the first and second chimeric polypeptides are associated by their Fc domains.

Embodiment I57 the method of any one of embodiments I44 to I56, wherein one or both of the first target binding domain and the second target binding domain specifically bind to a target selected from the group consisting of: CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNF alpha, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD, CD155, PDGF-DD, TGF-beta receptor II (TGF-. Beta.RII) ligand, TGF-. Beta.RIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, PDGF-DD receptor, receptor (SCF-DD receptor), receptor (receptor 3, MICP receptor) receptor of MIC-3, receptor-like receptor (CD-B) 122, receptor-3 of MIC-cell-like receptor (CD-3, receptor-B-receptor-3, receptor-B-receptor-4.

Embodiment I58 the method of any one of embodiments I44 to I56, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein.

Embodiment I59 the method of embodiment I58, wherein the soluble interleukin or cytokine protein is selected from the group consisting of: IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD and SCF.

Embodiment I60 the method of any one of embodiments I44 to I56, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor.

Embodiment I61 the method of embodiment I60, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII), soluble TGF-beta RIII, a soluble receptor for TNF alpha, a soluble receptor for IL-4, or a soluble receptor for IL-10.

Embodiment I62 the method of any one of embodiments I44 to I55, wherein the soluble tissue factor domain is a soluble human tissue factor domain that does not stimulate blood clotting.

Embodiment I63 the method of any one of embodiments I43 to I55, wherein the soluble tissue factor domain comprises or consists of a sequence from a wild-type soluble human tissue factor.

Sequence listing

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<120> methods of treating aging-related disorders

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<151> 2020-11-25

<150> 63/032,933

<151> 2020-06-01

<150> PCT/US2020/035598

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Leu Lys Lys Glu Pro Gly His Pro Trp Lys Trp Ser Asn Gly Lys Glu

145 150 155 160

Phe Asn Asn Trp Phe Asn Val Thr Gly Ser Asp Lys Cys Val Phe Leu

165 170 175

Lys Asn Thr Glu Val Ser Ser Met Glu Cys Glu Lys Asn Leu Tyr Trp

180 185 190

Ile Cys Asn Lys Pro Tyr Lys

195

<210> 4

<211> 600

<212> DNA

<213> Chile person

<400> 4

atgagctctg aaaattgttt cgtagcagag aacagctctt tgcatccgga gagtggacaa 60

gaaaatgatg ccaccagtcc ccatttctca acacgtcatg aagggtcctt ccaagttcct 120

gtcctgtgtg ctgtaatgaa tgtggtcttc atcaccattt taatcatagc tctcattgcc 180

ttatcagtgg gccaatacaa ttgtccaggc caatacacat tctcaatgcc atcagacagc 240

catgtttctt catgctctga ggactgggtt ggctaccaga ggaaatgcta ctttatttct 300

actgtgaaga ggagctggac ttcagcccaa aatgcttgtt ctgaacatgg tgctactctt 360

gctgtcattg attctgaaaa ggacatgaac tttctaaaac gatacgcagg tagagaggaa 420

cactgggttg gactgaaaaa ggaacctggt cacccatgga agtggtcaaa tggcaaagaa 480

tttaacaact ggttcaacgt tacagggtct gacaagtgtg tttttctgaa aaacacagag 540

gtcagcagca tggaatgtga gaagaattta tactggatat gtaacaaacc ttacaaataa 600

<210> 5

<211> 77

<212> PRT

<213> Chile person

<400> 5

Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala

1 5 10 15

Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly

20 25 30

Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn

35 40 45

Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys

50 55 60

Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn

65 70 75

<210> 6

<211> 387

<212> DNA

<213> Chile person

<400> 6

atgggaatcc aaggagggtc tgtcctgttc gggctgctgc tcgtcctggc tgtcttctgc 60

cattcaggtc atagcctgca gtgctacaac tgtcctaacc caactgctga ctgcaaaaca 120

gccgtcaatt gttcatctga ttttgatgcg tgtctcatta ccaaagctgg gttacaagtg 180

tataacaagt gttggaagtt tgagcattgc aatttcaacg acgtcacaac ccgcttgagg 240

gaaaatgagc taacgtacta ctgctgcaag aaggacctgt gtaactttaa cgaacagctt 300

gaaaatggtg ggacatcctt atcagagaaa acagttcttc tgctggtgac tccatttctg 360

gcagcagcct ggagccttca tccctaa 387

<210> 7

<211> 311

<212> PRT

<213> Chile person

<400> 7

Gln Ser Ser Leu Thr Pro Leu Met Val Asn Gly Ile Leu Gly Glu Ser

1 5 10 15

Val Thr Leu Pro Leu Glu Phe Pro Ala Gly Glu Lys Val Asn Phe Ile

20 25 30

Thr Trp Leu Phe Asn Glu Thr Ser Leu Ala Phe Ile Val Pro His Glu

35 40 45

Thr Lys Ser Pro Glu Ile His Val Thr Asn Pro Lys Gln Gly Lys Arg

50 55 60

Leu Asn Phe Thr Gln Ser Tyr Ser Leu Gln Leu Ser Asn Leu Lys Met

65 70 75 80

Glu Asp Thr Gly Ser Tyr Arg Ala Gln Ile Ser Thr Lys Thr Ser Ala

85 90 95

Lys Leu Ser Ser Tyr Thr Leu Arg Ile Leu Arg Gln Leu Arg Asn Ile

100 105 110

Gln Val Thr Asn His Ser Gln Leu Phe Gln Asn Met Thr Cys Glu Leu

115 120 125

His Leu Thr Cys Ser Val Glu Asp Ala Asp Asp Asn Val Ser Phe Arg

130 135 140

Trp Glu Ala Leu Gly Asn Thr Leu Ser Ser Gln Pro Asn Leu Thr Val

145 150 155 160

Ser Trp Asp Pro Arg Ile Ser Ser Glu Gln Asp Tyr Thr Cys Ile Ala

165 170 175

Glu Asn Ala Val Ser Asn Leu Ser Phe Ser Val Ser Ala Gln Lys Leu

180 185 190

Cys Glu Asp Val Lys Ile Gln Tyr Thr Asp Thr Lys Met Ile Leu Phe

195 200 205

Met Val Ser Gly Ile Cys Ile Val Phe Gly Phe Ile Ile Leu Leu Leu

210 215 220

Leu Val Leu Arg Lys Arg Arg Asp Ser Leu Ser Leu Ser Thr Gln Arg

225 230 235 240

Thr Gln Gly Pro Ala Glu Ser Ala Arg Asn Leu Glu Tyr Val Ser Val

245 250 255

Ser Pro Thr Asn Asn Thr Val Tyr Ala Ser Val Thr His Ser Asn Arg

260 265 270

Glu Thr Glu Ile Trp Thr Pro Arg Glu Asn Asp Thr Ile Thr Ile Tyr

275 280 285

Ser Thr Ile Asn His Ser Lys Glu Ser Lys Pro Thr Phe Ser Arg Ala

290 295 300

Thr Ala Leu Asp Asn Val Val

305 310

<210> 8

<211> 996

<212> DNA

<213> Chile person

<400> 8

atgttgtggc tgttccaatc gctcctgttt gtcttctgct ttggcccagg gaatgtagtt 60

tcacaaagca gcttaacccc attgatggtg aacgggattc tgggggagtc agtaactctt 120

cccctggagt ttcctgcagg agagaaggtc aacttcatca cttggctttt caatgaaaca 180

tctcttgcct tcatagtacc ccatgaaacc aaaagtccag aaatccacgt gactaatccg 240

aaacagggaa agcgactgaa cttcacccag tcctactccc tgcaactcag caacctgaag 300

atggaagaca caggctctta cagagcccag atatccacaa agacctctgc aaagctgtcc 360

agttacactc tgaggatatt aagacaactg aggaacatac aagttaccaa tcacagtcag 420

ctatttcaga atatgacctg tgagctccat ctgacttgct ctgtggagga tgcagatgac 480

aatgtctcat tcagatggga ggccttggga aacacacttt caagtcagcc aaacctcact 540

gtctcctggg accccaggat ttccagtgaa caggactaca cctgcatagc agagaatgct 600

gtcagtaatt tatccttctc tgtctctgcc cagaagcttt gcgaagatgt taaaattcaa 660

tatacagata ccaaaatgat tctgtttatg gtttctggga tatgcatagt cttcggtttc 720

atcatactgc tgttacttgt tttgaggaaa agaagagatt ccctatcttt gtctactcag 780

cgaacacagg gccccgagtc cgcaaggaac ctagagtatg tttcagtgtc tccaacgaac 840

aacactgtgt atgcttcagt cactcattca aacagggaaa cagaaatctg gacacctaga 900

gaaaatgata ctatcacaat ttactccaca attaatcatt ccaaagagag taaacccact 960

ttttccaggg caactgccct tgacaatgtc gtgtaa 996

<210> 9

<211> 231

<212> PRT

<213> Chile person

<400> 9

Met Gln Asp Glu Glu Arg Tyr Met Thr Leu Asn Val Gln Ser Lys Lys

1 5 10 15

Arg Ser Ser Ala Gln Thr Ser Gln Leu Thr Phe Lys Asp Tyr Ser Val

20 25 30

Thr Leu His Trp Tyr Lys Ile Leu Leu Gly Ile Ser Gly Thr Val Asn

35 40 45

Gly Ile Leu Thr Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Ser Gln

50 55 60

Gly Val Leu Leu Lys Cys Gln Lys Gly Ser Cys Ser Asn Ala Thr Gln

65 70 75 80

Tyr Glu Asp Thr Gly Asp Leu Lys Val Asn Asn Gly Thr Arg Arg Asn

85 90 95

Ile Ser Asn Lys Asp Leu Cys Ala Ser Arg Ser Ala Asp Gln Thr Val

100 105 110

Leu Cys Gln Ser Glu Trp Leu Lys Tyr Gln Gly Lys Cys Tyr Trp Phe

115 120 125

Ser Asn Glu Met Lys Ser Trp Ser Asp Ser Tyr Val Tyr Cys Leu Glu

130 135 140

Arg Lys Ser His Leu Leu Ile Ile His Asp Gln Leu Glu Met Ala Phe

145 150 155 160

Ile Gln Lys Asn Leu Arg Gln Leu Asn Tyr Val Trp Ile Gly Leu Asn

165 170 175

Phe Thr Ser Leu Lys Met Thr Trp Thr Trp Val Asp Gly Ser Pro Ile

180 185 190

Asp Ser Lys Ile Phe Phe Ile Lys Gly Pro Ala Lys Glu Asn Ser Cys

195 200 205

Ala Ala Ile Lys Glu Ser Lys Ile Phe Ser Glu Thr Cys Ser Ser Val

210 215 220

Phe Lys Trp Ile Cys Gln Tyr

225 230

<210> 10

<211> 546

<212> DNA

<213> Chile person

<400> 10

atgcaagatg aagaaagata catgacattg aatgtacagt caaagaaaag gagttctgcc 60

caaacatctc aacttacatt taaagattat tcagtgacgt tgcactggta taaaatctta 120

ctgggaatat ctggaaccgt gaatggtatt ctcactttga ctttgatctc cttgatcctg 180

ttggtactat gccaatcaga atggctcaaa taccaaggga agtgttattg gttctctaat 240

gagatgaaaa gctggagtga cagttatgtg tattgtttgg aaagaaaatc tcatctacta 300

atcatacatg accaacttga aatggctttt atacagaaaa acctaagaca attaaactac 360

gtatggattg ggcttaactt tacctccttg aaaatgacat ggacttgggt ggatggttct 420

ccaatagatt caaagatatt cttcataaag ggaccagcta aagaaaacag ctgtgctgcc 480

attaaggaaa gcaaaatttt ctctgaaacc tgcagcagtg ttttcaaatg gatttgtcag 540

tattag 546

<210> 11

<211> 318

<212> PRT

<213> Chile person

<400> 11

Glu Glu Val Leu Trp His Thr Ser Val Pro Phe Ala Glu Asn Met Ser

1 5 10 15

Leu Glu Cys Val Tyr Pro Ser Met Gly Ile Leu Thr Gln Val Glu Trp

20 25 30

Phe Lys Ile Gly Thr Gln Gln Asp Ser Ile Ala Ile Phe Ser Pro Thr

35 40 45

His Gly Met Val Ile Arg Lys Pro Tyr Ala Glu Arg Val Tyr Phe Leu

50 55 60

Asn Ser Thr Met Ala Ser Asn Asn Met Thr Leu Phe Phe Arg Asn Ala

65 70 75 80

Ser Glu Asp Asp Val Gly Tyr Tyr Ser Cys Ser Leu Tyr Thr Tyr Pro

85 90 95

Gln Gly Thr Trp Gln Lys Val Ile Gln Val Val Gln Ser Asp Ser Phe

100 105 110

Glu Ala Ala Val Pro Ser Asn Ser His Ile Val Ser Glu Pro Gly Lys

115 120 125

Asn Val Thr Leu Thr Cys Gln Pro Gln Met Thr Trp Pro Val Gln Ala

130 135 140

Val Arg Trp Glu Lys Ile Gln Pro Arg Gln Ile Asp Leu Leu Thr Tyr

145 150 155 160

Cys Asn Leu Val His Gly Arg Asn Phe Thr Ser Lys Phe Pro Arg Gln

165 170 175

Ile Val Ser Asn Cys Ser His Gly Arg Trp Ser Val Ile Val Ile Pro

180 185 190

Asp Val Thr Val Ser Asp Ser Gly Leu Tyr Arg Cys Tyr Leu Gln Ala

195 200 205

Ser Ala Gly Glu Asn Glu Thr Phe Val Met Arg Leu Thr Val Ala Glu

210 215 220

Gly Lys Thr Asp Asn Gln Tyr Thr Leu Phe Val Ala Gly Gly Thr Val

225 230 235 240

Leu Leu Leu Leu Phe Val Ile Ser Ile Thr Thr Ile Ile Val Ile Phe

245 250 255

Leu Asn Arg Arg Arg Arg Arg Glu Arg Arg Asp Leu Phe Thr Glu Ser

260 265 270

Trp Asp Thr Gln Lys Ala Pro Asn Asn Tyr Arg Ser Pro Ile Ser Thr

275 280 285

Ser Gln Pro Thr Asn Gln Ser Met Asp Asp Thr Arg Glu Asp Ile Tyr

290 295 300

Val Asn Tyr Pro Thr Phe Ser Arg Arg Pro Lys Thr Arg Val

305 310 315

<210> 12

<211> 1011

<212> DNA

<213> Chile person

<400> 12

atggattatc ctactttact tttggctctt cttcatgtat acagagctct atgtgaagag 60

gtgctttggc atacatcagt tccctttgcc gagaacatgt ctctagaatg tgtgtatcca 120

tcaatgggca tcttaacaca ggtggagtgg ttcaagatcg ggacccagca ggattccata 180

gccattttca gccctactca tggcatggtc ataaggaagc cctatgctga gagggtttac 240

tttttgaatt caacgatggc ttccaataac atgactcttt tctttcggaa tgcctctgaa 300

gatgatgttg gctactattc ctgctctctt tacacttacc cacagggaac ttggcagaag 360

gtgatacagg tggttcagtc agatagtttt gaggcagctg tgccatcaaa tagccacatt 420

gtttcggaac ctggaaagaa tgtcacactc acttgtcagc ctcagatgac gtggcctgtg 480

caggcagtga ggtgggaaaa gatccagccc cgtcagatcg acctcttaac ttactgcaac 540

ttggtccatg gcagaaattt cacctccaag ttcccaagac aaatagtgag caactgcagc 600

cacggaaggt ggagcgtcat cgtcatcccc gatgtcacag tctcagactc ggggctttac 660

cgctgctact tgcaggccag cgcaggagaa aacgaaacct tcgtgatgag attgactgta 720

gccgagggta aaaccgataa ccaatatacc ctctttgtgg ctggagggac agttttattg 780

ttgttgtttg ttatctcaat taccaccatc attgtcattt tccttaacag aaggagaagg 840

agagagagaa gagatctatt tacagagtcc tgggatacac agaaggcacc caataactat 900

agaagtccca tctctaccag tcaacctacc aatcaatcca tggatgatac aagagaggat 960

atttatgtca actatccaac cttctctcgc agaccaaaga ctagagttta a 1011

<210> 13

<211> 347

<212> PRT

<213> Chile person

<400> 13

Gly Lys Gly Cys Gln Gly Ser Ala Asp His Val Val Ser Ile Ser Gly

1 5 10 15

Val Pro Leu Gln Leu Gln Pro Asn Ser Ile Gln Thr Lys Val Asp Ser

20 25 30

Ile Ala Trp Lys Lys Leu Leu Pro Ser Gln Asn Gly Phe His His Ile

35 40 45

Leu Lys Trp Glu Asn Gly Ser Leu Pro Ser Asn Thr Ser Asn Asp Arg

50 55 60

Phe Ser Phe Ile Val Lys Asn Leu Ser Leu Leu Ile Lys Ala Ala Gln

65 70 75 80

Gln Gln Asp Ser Gly Leu Tyr Cys Leu Glu Val Thr Ser Ile Ser Gly

85 90 95

Lys Val Gln Thr Ala Thr Phe Gln Val Phe Val Phe Asp Lys Val Glu

100 105 110

Lys Pro Arg Leu Gln Gly Gln Gly Lys Ile Leu Asp Arg Gly Arg Cys

115 120 125

Gln Val Ala Leu Ser Cys Leu Val Ser Arg Asp Gly Asn Val Ser Tyr

130 135 140

Ala Trp Tyr Arg Gly Ser Lys Leu Ile Gln Thr Ala Gly Asn Leu Thr

145 150 155 160

Tyr Leu Asp Glu Glu Val Asp Ile Asn Gly Thr His Thr Tyr Thr Cys

165 170 175

Asn Val Ser Asn Pro Val Ser Trp Glu Ser His Thr Leu Asn Leu Thr

180 185 190

Gln Asp Cys Gln Asn Ala His Gln Glu Phe Arg Phe Trp Pro Phe Leu

195 200 205

Val Ile Ile Val Ile Leu Ser Ala Leu Phe Leu Gly Thr Leu Ala Cys

210 215 220

Phe Cys Val Trp Arg Arg Lys Arg Lys Glu Lys Gln Ser Glu Thr Ser

225 230 235 240

Pro Lys Glu Phe Leu Thr Ile Tyr Glu Asp Val Lys Asp Leu Lys Thr

245 250 255

Arg Arg Asn His Glu Gln Glu Gln Thr Phe Pro Gly Gly Gly Ser Thr

260 265 270

Ile Tyr Ser Met Ile Gln Ser Gln Ser Ser Ala Pro Thr Ser Gln Glu

275 280 285

Pro Ala Tyr Thr Leu Tyr Ser Leu Ile Gln Pro Ser Arg Lys Ser Gly

290 295 300

Ser Arg Lys Arg Asn His Ser Pro Ser Phe Asn Ser Thr Ile Tyr Glu

305 310 315 320

Val Ile Gly Lys Ser Gln Pro Lys Ala Gln Asn Pro Ala Arg Leu Ser

325 330 335

Arg Lys Glu Leu Glu Asn Phe Asp Val Tyr Ser

340 345

<210> 14

<211> 1098

<212> DNA

<213> Chile person

<400> 14

atgctggggc aagtggtcac cctcatactc ctcctgctcc tcaaggtgta tcagggcaaa 60

ggatgccagg gatcagctga ccatgtggtt agcatctcgg gagtgcctct tcagttacaa 120

ccaaacagca tacagacgaa ggttgacagc attgcatgga agaagttgct gccctcacaa 180

aatggatttc atcacatatt gaagtgggag aatggctctt tgccttccaa tacttccaat 240

gatagattca gttttatagt caagaacttg agtcttctca tcaaggcagc tcagcagcag 300

gacagtggcc tctactgcct ggaggtcacc agtatatctg gaaaagttca gacagccacg 360

ttccaggttt ttgtatttga taaagttgag aaaccccgcc tacaggggca ggggaagatc 420

ctggacagag ggagatgcca agtggctctg tcttgcttgg tctccaggga tggcaatgtg 480

tcctatgctt ggtacagagg gagcaagctg atccagacag cagggaacct cacctacctg 540

gacgaggagg ttgacattaa tggcactcac acatatacct gcaatgtcag caatcctgtt 600

agctgggaaa gccacaccct gaatctcact caggactgtc agaatgccca tcaggaattc 660

agattttggc cgtttttggt gatcatcgtg attctaagcg cactgttcct tggcaccctt 720

gcctgcttct gtgtgtggag gagaaagagg aaggagaagc agtcagagac cagtcccaag 780

gaatttttga caatttacga agatgtcaag gatctgaaaa ccaggagaaa tcacgagcag 840

gagcagactt ttcctggagg ggggagcacc atctactcta tgatccagtc ccagtcttct 900

gctcccacgt cacaagaacc tgcatataca ttatattcat taattcagcc ttccaggaag 960

tctggatcca ggaagaggaa ccacagccct tccttcaata gcactatcta tgaagtgatt 1020

ggaaagagtc aacctaaagc ccagaaccct gctcgattga gccgcaaaga gctggagaac 1080

tttgatgttt attcctag 1098

<210> 15

<211> 183

<212> PRT

<213> Chile person

<400> 15

Leu Trp Val Ser Gln Pro Pro Glu Ile Arg Thr Leu Glu Gly Ser Ser

1 5 10 15

Ala Phe Leu Pro Cys Ser Phe Asn Ala Ser Gln Gly Arg Leu Ala Ile

20 25 30

Gly Ser Val Thr Trp Phe Arg Asp Glu Val Val Pro Gly Lys Glu Val

35 40 45

Arg Asn Gly Thr Pro Glu Phe Arg Gly Arg Leu Ala Pro Leu Ala Ser

50 55 60

Ser Arg Phe Leu His Asp His Gln Ala Glu Leu His Ile Arg Asp Val

65 70 75 80

Arg Gly His Asp Ala Ser Ile Tyr Val Cys Arg Val Glu Val Leu Gly

85 90 95

Leu Gly Val Gly Thr Gly Asn Gly Thr Arg Leu Val Val Glu Lys Glu

100 105 110

His Pro Gln Leu Gly Ala Gly Thr Val Leu Leu Leu Arg Ala Gly Phe

115 120 125

Tyr Ala Val Ser Phe Leu Ser Val Ala Val Gly Ser Thr Val Tyr Tyr

130 135 140

Gln Gly Lys Cys Leu Thr Trp Lys Gly Pro Arg Arg Gln Leu Pro Ala

145 150 155 160

Val Val Pro Ala Pro Leu Pro Pro Pro Cys Gly Ser Ser Ala His Leu

165 170 175

Leu Pro Pro Val Pro Gly Gly

180

<210> 16

<211> 573

<212> DNA

<213> Chile person

<400> 16

atggcctgga tgctgttgct catcttgatc atggtccatc caggatcctg tgctctctgg 60

gtgtcccagc cccctgagat tcgtaccctg gaaggatcct ctgccttcct gccctgctcc 120

ttcaatgcca gccaagggag actggccatt ggctccgtca cgtggttccg agatgaggtg 180

gttccaggga aggaggtgag gaatggaacc ccagagttca ggggccgcct ggccccactt 240

gcttcttccc gtttcctcca tgaccaccag gctgagctgc acatccggga cgtgcgaggc 300

catgacgcca gcatctacgt gtgcagagtg gaggtgctgg gccttggtgt cgggacaggg 360

aatgggactc ggctggtggt ggagaaagaa catcctcagc taggggctgg tacagtcctc 420

ctccttcggg ctggattcta tgctgtcagc tttctctctg tggccgtggg cagcaccgtc 480

tattaccagg gcaaatgcca ctgtcacatg ggaacacact gccactcctc agatgggccc 540

cgaggagtga ttccagagcc cagatgtccc tag 573

<210> 17

<211> 255

<212> PRT

<213> Chile person

<400> 17

Gln Ser Lys Ala Gln Val Leu Gln Ser Val Ala Gly Gln Thr Leu Thr

1 5 10 15

Val Arg Cys Gln Tyr Pro Pro Thr Gly Ser Leu Tyr Glu Lys Lys Gly

20 25 30

Trp Cys Lys Glu Ala Ser Ala Leu Val Cys Ile Arg Leu Val Thr Ser

35 40 45

Ser Lys Pro Arg Thr Met Ala Trp Thr Ser Arg Phe Thr Ile Trp Asp

50 55 60

Asp Pro Asp Ala Gly Phe Phe Thr Val Thr Met Thr Asp Leu Arg Glu

65 70 75 80

Glu Asp Ser Gly His Tyr Trp Cys Arg Ile Tyr Arg Pro Ser Asp Asn

85 90 95

Ser Val Ser Lys Ser Val Arg Phe Tyr Leu Val Val Ser Pro Ala Ser

100 105 110

Ala Ser Thr Gln Thr Ser Trp Thr Pro Arg Asp Leu Val Ser Ser Gln

115 120 125

Thr Gln Thr Gln Ser Cys Val Pro Pro Thr Ala Gly Ala Arg Gln Ala

130 135 140

Pro Glu Ser Pro Ser Thr Ile Pro Val Pro Ser Gln Pro Gln Asn Ser

145 150 155 160

Thr Leu Arg Pro Gly Pro Ala Ala Pro Ile Ala Leu Val Pro Val Phe

165 170 175

Cys Gly Leu Leu Val Ala Lys Ser Leu Val Leu Ser Ala Leu Leu Val

180 185 190

Trp Trp Gly Asp Ile Trp Trp Lys Thr Met Met Glu Leu Arg Ser Leu

195 200 205

Asp Thr Gln Lys Ala Thr Cys His Leu Gln Gln Val Thr Asp Leu Pro

210 215 220

Trp Thr Ser Val Ser Ser Pro Val Glu Arg Glu Ile Leu Tyr His Thr

225 230 235 240

Val Ala Arg Thr Lys Ile Ser Asp Asp Asp Asp Glu His Thr Leu

245 250 255

<210> 18

<211> 777

<212> DNA

<213> Chile person

<400> 18

atggcctggc gagccctaca cccactgcta ctgctgctgc tgctgttccc aggctctcag 60

gcacaatcca aggctcaggt acttcaaagt gtggcagggc agacgctaac cgtgagatgc 120

cagtacccgc ccacgggcag tctctacgag aagaaaggct ggtgtaagga ggcttcagca 180

cttgtgtgca tcaggttagt caccagctcc aagcccagga cgatggcttg gacctctcga 240

ttcacaatct gggacgaccc tgatgctggc ttcttcactg tcaccatgac tgatctgaga 300

gaggaagact caggacatta ctggtgtaga atctaccgcc cttctgacaa ctctgtctct 360

aagtccgtca gattctatct ggtggtatct ccagcctctg cctccacaca gacctcctgg 420

actccccgcg acctggtctc ttcacagacc cagacccaga gctgtgtgcc tcccactgca 480

ggagccagac aagcccctga gtctccatct accatccctg tcccttcaca gccacagaac 540

tccacgctcc gccctggccc tgcagccccc attgccctgg tgcctgtgtt ctgtggactc 600

ctcgtagcca agagcctggt gctgtcagcc ctgctcgtct ggtgggtttt aaggaatcgg 660

cacatgcagc atcaagggag gtctctgctg cacccagctc agcccaggcc ccaggcccat 720

agacacttcc cactgagcca cagggcacca ggggggacat atggtggaaa accatga 777

<210> 19

<211> 283

<212> PRT

<213> Chile person

<400> 19

Gln Gln Gln Thr Leu Pro Lys Pro Phe Ile Trp Ala Glu Pro His Phe

1 5 10 15

Met Val Pro Lys Glu Lys Gln Val Thr Ile Cys Cys Gln Gly Asn Tyr

20 25 30

Gly Ala Val Glu Tyr Gln Leu His Phe Glu Gly Ser Leu Phe Ala Val

35 40 45

Asp Arg Pro Lys Pro Pro Glu Arg Ile Asn Lys Val Gln Phe Tyr Ile

50 55 60

Pro Asp Met Asn Ser Arg Met Ala Gly Gln Tyr Ser Cys Ile Tyr Arg

65 70 75 80

Val Gly Glu Leu Trp Ser Glu Pro Ser Asn Leu Leu Asp Leu Val Val

85 90 95

Thr Glu Met Tyr Asp Thr Pro Thr Leu Ser Val His Pro Gly Pro Glu

100 105 110

Val Ile Ser Gly Glu Lys Val Thr Phe Tyr Cys Arg Leu Asp Thr Ala

115 120 125

Thr Ser Met Phe Leu Leu Leu Lys Glu Gly Arg Ser Ser His Val Gln

130 135 140

Arg Gly Tyr Gly Lys Val Gln Ala Glu Phe Pro Leu Gly Pro Val Thr

145 150 155 160

Thr Ala His Arg Gly Thr Tyr Arg Cys Phe Gly Ser Tyr Asn Asn His

165 170 175

Ala Trp Ser Phe Pro Ser Glu Pro Val Lys Leu Leu Val Thr Gly Asp

180 185 190

Ile Glu Asn Thr Ser Leu Ala Pro Glu Asp Pro Thr Phe Pro Ala Asp

195 200 205

Thr Trp Gly Thr Tyr Leu Leu Thr Thr Glu Thr Gly Leu Gln Lys Asp

210 215 220

His Ala Leu Trp Asp His Thr Ala Gln Asn Leu Leu Arg Met Gly Leu

225 230 235 240

Ala Phe Leu Val Leu Val Ala Leu Val Trp Phe Leu Val Glu Asp Trp

245 250 255

Leu Ser Arg Lys Arg Thr Arg Glu Arg Ala Ser Arg Ala Ser Thr Trp

260 265 270

Glu Gly Arg Arg Arg Leu Asn Thr Gln Thr Leu

275 280

<210> 20

<211> 777

<212> DNA

<213> Chile person

<400> 20

atggcctggc gagccctaca cccactgcta ctgctgctgc tgctgttccc aggctctcag 60

gcacaatcca aggctcaggt acttcaaagt gtggcagggc agacgctaac cgtgagatgc 120

cagtacccgc ccacgggcag tctctacgag aagaaaggct ggtgtaagga ggcttcagca 180

cttgtgtgca tcaggttagt caccagctcc aagcccagga cgatggcttg gacctctcga 240

ttcacaatct gggacgaccc tgatgctggc ttcttcactg tcaccatgac tgatctgaga 300

gaggaagact caggacatta ctggtgtaga atctaccgcc cttctgacaa ctctgtctct 360

aagtccgtca gattctatct ggtggtatct ccagcctctg cctccacaca gacctcctgg 420

actccccgcg acctggtctc ttcacagacc cagacccaga gctgtgtgcc tcccactgca 480

ggagccagac aagcccctga gtctccatct accatccctg tcccttcaca gccacagaac 540

tccacgctcc gccctggccc tgcagccccc attgccctgg tgcctgtgtt ctgtggactc 600

ctcgtagcca agagcctggt gctgtcagcc ctgctcgtct ggtgggtttt aaggaatcgg 660

cacatgcagc atcaagggag gtctctgctg cacccagctc agcccaggcc ccaggcccat 720

agacacttcc cactgagcca cagggcacca ggggggacat atggtggaaa accatga 777

<210> 21

<211> 216

<212> PRT

<213> Chile person

<400> 21

Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp Glu Met Ser

1 5 10 15

Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp Phe Ser Thr

20 25 30

Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys Cys Arg Glu

35 40 45

Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala Met Gly

50 55 60

Ile Arg Phe Ile Ile Met Val Ala Ile Trp Ser Ala Val Phe Leu Asn

65 70 75 80

Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys

85 90 95

Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln

100 105 110

Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met

115 120 125

Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp

130 135 140

Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile

145 150 155 160

Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro

165 170 175

Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr

180 185 190

Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr

195 200 205

Tyr Ile Cys Met Gln Arg Thr Val

210 215

<210> 22

<211> 651

<212> DNA

<213> Chile person

<400> 22

atggggtgga ttcgtggtcg gaggtctcga cacagctggg agatgagtga atttcataat 60

tataacttgg atctgaagaa gagtgatttt tcaacacgat ggcaaaagca aagatgtcca 120

gtagtcaaaa gcaaatgtag agaaaatgca tctccatttt ttttctgctg cttcatcgct 180

gtagccatgg gaatccgttt cattattatg gtaacaatat ggagtgctgt attcctaaac 240

tcattattca accaagaagt tcaaattccc ttgaccgaaa gttactgtgg cccatgtcct 300

aaaaactgga tatgttacaa aaataactgc taccaatttt ttgatgagag taaaaactgg 360

tatgagagcc aggcttcttg tatgtctcaa aatgccagcc ttctgaaagt atacagcaaa 420

gaggaccagg atttacttaa actggtgaag tcatatcatt ggatgggact agtacacatt 480

ccaacaaatg gatcttggca gtgggaagat ggctccattc tctcacccaa cctactaaca 540

ataattgaaa tgcagaaggg agactgtgca ctctatgcct cgagctttaa aggctatata 600

gaaaactgtt caactccaaa tacgtacatc tgcatgcaaa ggactgtgta a 651

<210> 23

<211> 359

<212> PRT

<213> Chile person

<400> 23

Met Ala Glu Gly Thr Leu Trp Gln Ile Leu Cys Val Ser Ser Asp Ala

1 5 10 15

Gln Pro Gln Thr Phe Glu Gly Val Lys Gly Ala Asp Pro Pro Thr Leu

20 25 30

Pro Pro Gly Ser Phe Leu Pro Gly Pro Val Leu Trp Trp Gly Ser Leu

35 40 45

Ala Arg Leu Gln Thr Glu Lys Ser Asp Glu Val Ser Arg Lys Gly Asn

50 55 60

Trp Trp Val Thr Glu Met Gly Gly Gly Ala Gly Glu Arg Leu Phe Thr

65 70 75 80

Ser Ser Cys Leu Val Gly Leu Val Pro Leu Gly Leu Arg Ile Ser Leu

85 90 95

Val Thr Cys Pro Leu Gln Cys Gly Ile Met Trp Gln Leu Leu Leu Pro

100 105 110

Thr Ala Leu Leu Leu Leu Val Ser Ala Gly Met Arg Thr Glu Asp Leu

115 120 125

Pro Lys Ala Val Val Phe Leu Glu Pro Gln Trp Tyr Arg Val Leu Glu

130 135 140

Lys Asp Ser Val Thr Leu Lys Cys Gln Gly Ala Tyr Ser Pro Glu Asp

145 150 155 160

Asn Ser Thr Gln Trp Phe His Asn Glu Ser Leu Ile Ser Ser Gln Ala

165 170 175

Ser Ser Tyr Phe Ile Asp Ala Ala Thr Val Asp Asp Ser Gly Glu Tyr

180 185 190

Arg Cys Gln Thr Asn Leu Ser Thr Leu Ser Asp Pro Val Gln Leu Glu

195 200 205

Val His Ile Gly Trp Leu Leu Leu Gln Ala Pro Arg Trp Val Phe Lys

210 215 220

Glu Glu Asp Pro Ile His Leu Arg Cys His Ser Trp Lys Asn Thr Ala

225 230 235 240

Leu His Lys Val Thr Tyr Leu Gln Asn Gly Lys Gly Arg Lys Tyr Phe

245 250 255

His His Asn Ser Asp Phe Tyr Ile Pro Lys Ala Thr Leu Lys Asp Ser

260 265 270

Gly Ser Tyr Phe Cys Arg Gly Leu Phe Gly Ser Lys Asn Val Ser Ser

275 280 285

Glu Thr Val Asn Ile Thr Ile Thr Gln Gly Leu Ala Val Ser Thr Ile

290 295 300

Ser Ser Phe Phe Pro Pro Gly Tyr Gln Val Ser Phe Cys Leu Val Met

305 310 315 320

Val Leu Leu Phe Ala Val Asp Thr Gly Leu Tyr Phe Ser Val Lys Thr

325 330 335

Asn Ile Arg Ser Ser Thr Arg Asp Trp Lys Asp His Lys Phe Lys Trp

340 345 350

Arg Lys Asp Pro Gln Asp Lys

355

<210> 24

<211> 1080

<212> DNA

<213> Chile person

<400> 24

atggctgagg gcacactctg gcagattctg tgtgtgtcct cagatgctca gccacagacc 60

tttgagggag taaagggggc agacccaccc accttgcctc caggctcttt ccttcctggt 120

cctgttctat ggtggggctc ccttgccaga cttcagactg agaagtcaga tgaagtttca 180

agaaaaggaa attggtgggt gacagagatg ggtggagggg ctggggaaag gctgtttact 240

tcctcctgtc tagtcggttt ggtcccttta gggctccgga tatctttggt gacttgtcca 300

ctccagtgtg gcatcatgtg gcagctgctc ctcccaactg ctctgctact tctagtttca 360

gctggcatgc ggactgaaga tctcccaaag gctgtggtgt tcctggagcc tcaatggtac 420

agggtgctcg agaaggacag tgtgactctg aagtgccagg gagcctactc ccctgaggac 480

aattccacac agtggtttca caatgagagc ctcatctcaa gccaggcctc gagctacttc 540

attgacgctg ccacagtcga cgacagtgga gagtacaggt gccagacaaa cctctccacc 600

ctcagtgacc cggtgcagct agaagtccat atcggctggc tgttgctcca ggcccctcgg 660

tgggtgttca aggaggaaga ccctattcac ctgaggtgtc acagctggaa gaacactgct 720

ctgcataagg tcacatattt acagaatggc aaaggcagga agtattttca tcataattct 780

gacttctaca ttccaaaagc cacactcaaa gacagcggct cctacttctg cagggggctt 840

tttgggagta aaaatgtgtc ttcagagact gtgaacatca ccatcactca aggtttggca 900

gtgtcaacca tctcatcatt ctttccacct gggtaccaag tctctttctg cttggtgatg 960

gtactccttt ttgcagtgga cacaggacta tatttctctg tgaagacaaa cattcgaagc 1020

tcaacaagag actggaagga ccataaattt aaatggagaa aggaccctca agacaaatga 1080

<210> 25

<211> 233

<212> PRT

<213> Chile person

<400> 25

Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala

1 5 10 15

Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro

20 25 30

Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln

35 40 45

Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu

50 55 60

Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr

65 70 75 80

Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu

85 90 95

Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln

100 105 110

Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys

115 120 125

His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn

130 135 140

Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro

145 150 155 160

Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val

165 170 175

Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln

180 185 190

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln

195 200 205

Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly

210 215 220

Leu Tyr Phe Ser Val Lys Thr Asn Ile

225 230

<210> 26

<211> 702

<212> DNA

<213> Chile person

<400> 26

atgtggcagc tgctcctccc aactgctctg ctacttctag tttcagctgg catgcggact 60

gaagatctcc caaaggctgt ggtgttcctg gagcctcaat ggtacagcgt gcttgagaag 120

gacagtgtga ctctgaagtg ccagggagcc tactcccctg aggacaattc cacacagtgg 180

tttcacaatg agaacctcat ctcaagccag gcctcgagct acttcattga cgctgccaca 240

gtcaacgaca gtggagagta caggtgccag acaaacctct ccaccctcag tgacccggtg 300

cagctagaag tccatatcgg ctggctgttg ctccaggccc ctcggtgggt gttcaaggag 360

gaagacccta ttcacctgag gtgtcacagc tggaagaaca ctgctctgca taaggtcaca 420

tatttacaga atggcaaaga caggaagtat tttcatcata attctgactt ccacattcca 480

aaagccacac tcaaagatag cggctcctac ttctgcaggg ggcttgttgg gagtaaaaat 540

gtgtcttcag agactgtgaa catcaccatc actcaaggtt tggcagtgtc aaccatctca 600

tcattctctc cacctgggta ccaagtctct ttctgcttgg tgatggtact cctttttgca 660

gtggacacag gactatattt ctctgtgaag acaaacattt ga 702

<210> 27

<211> 304

<212> PRT

<213> Chile person

<400> 27

Met Ser Leu Thr Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Gln Gly Ala Trp Pro His Glu Gly Val His Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Arg Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Met Phe Glu His Phe Leu Leu His Arg Glu Gly

50 55 60

Met Phe Asn Asp Thr Leu Arg Leu Ile Gly Glu His His Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Ser Arg Met Lys Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Thr His Ser Pro Tyr Gln Val Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Ile Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser

130 135 140

Val Thr Leu Ser Cys Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Arg Glu Gly Glu Ala His Glu Arg Arg Leu Pro Ala Gly Thr Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asn Phe Pro Leu Gly Pro Ala Thr His

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Lys Ser Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Glu Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Lys Ile Pro

245 250 255

Phe Thr Ile Leu Leu Phe Phe Leu Leu His Arg Trp Cys Ser Asp Lys

260 265 270

Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val

275 280 285

Asn Ser Glu Asp Ser Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala

290 295 300

<210> 28

<211> 915

<212> DNA

<213> Chile person

<400> 28

atgtcgctca cggtcgtcag catggcgtgt gttgggttct tcttgctgca gggggcctgg 60

ccacatgagg gagtccacag aaaaccttcc ctcctggccc acccaggtcg cctggtgaaa 120

tcagaagaga cagtcatcct gcaatgttgg tcagatgtca tgtttgaaca cttccttctg 180

cacagagagg ggatgtttaa cgacactttg cgcctcattg gagaacacca tgatggggtc 240

tccaaggcca acttctccat cagtcgcatg aagcaagacc tggcagggac ctacagatgc 300

tacggttctg ttactcactc cccctatcag ttgtcagctc ccagtgaccc tctggacatc 360

gtgatcatag gtctatatga gaaaccttct ctctcagccc agccgggccc cacggttctg 420

gcaggagaga atgtgacctt gtcctgcagc tcccggagct cctatgacat gtaccatcta 480

tccagggaag gggaggccca tgaacgtagg ctccctgcag ggaccaaggt caacggaaca 540

ttccaggcca actttcctct gggccctgcc acccatggag ggacctacag atgcttcggc 600

tctttccgtg actctccata cgagtggtca aagtcaagtg acccactgct tgtttctgtc 660

acaggaaacc cttcaaatag ttggccttca cccactgaac caagctccga aaccggtaac 720

cccagacacc tacatgttct gattgggacc tcagtggtca aaatcccttt caccatcctc 780

ctcttctttc tccttcatcg ctggtgctcc gacaaaaaaa atgctgctgt aatggaccaa 840

gagcctgcag ggaacagaac agtgaacagc gaggattctg atgaacaaga ccatcaggag 900

gtgtcatacg cataa 915

<210> 29

<211> 304

<212> PRT

<213> Chile person

<400> 29

Met Ser Leu Met Val Val Ser Met Val Cys Val Gly Phe Phe Leu Leu

1 5 10 15

Gln Gly Ala Trp Pro His Glu Gly Val His Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Pro Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Arg Phe Glu His Phe Leu Leu His Arg Glu Gly

50 55 60

Lys Tyr Lys Asp Thr Leu His Leu Ile Gly Glu His His Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Gly Pro Met Met Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser

130 135 140

Val Thr Leu Ser Cys Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Arg Glu Gly Glu Ala His Glu Arg Arg Phe Ser Ala Gly Pro Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Asn Ser Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Lys Ile Pro

245 250 255

Phe Thr Ile Leu Leu Phe Phe Leu Leu His Arg Trp Cys Ser Asn Lys

260 265 270

Lys Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asn Arg Thr Val

275 280 285

Asn Ser Glu Asp Ser Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala

290 295 300

<210> 30

<211> 1026

<212> DNA

<213> Chile person

<400> 30

atgtcgctca tggtcgtcag catggcgtgt gttgggttct tcttgctgca gggggcctgg 60

ccacatgagg gagtccacag aaaaccttcc ctcctggccc acccaggtcc cctggtgaaa 120

tcagaagaga cagtcatcct gcaatgttgg tcagatgtca ggtttgagca cttccttctg 180

cacagagagg ggaagtataa ggacactttg cacctcattg gagagcacca tgatggggtc 240

tccaaggcca acttctccat cggtcccatg atgcaagacc ttgcagggac ctacagatgc 300

tacggttctg ttactcactc cccctatcag ttgtcagctc ccagtgaccc tctggacatc 360

gtcatcacag gtctatatga gaaaccttct ctctcagccc agccgggccc cacggttttg 420

gcaggagaga gcgtgacctt gtcctgcagc tcccggagct cctatgacat gtaccatcta 480

tccagggagg gggaggccca tgaacgtagg ttctctgcag ggcccaaggt caacggaaca 540

ttccaggccg actttcctct gggccctgcc acccacggag gaacctacag atgcttcggc 600

tctttccgtg actctcccta tgagtggtca aactcgagtg acccactgct tgtttctgtc 660

acaggaaacc cttcaaatag ttggccttca cccactgaac caagctccaa aaccggtaac 720

cccagacacc tgcatgttct gattgggacc tcagtggtca aaatcccttt caccatcctc 780

ctcttctttc tccttcatcg ctggtgctcc aacaaaaaaa atgctgctgt aatggaccaa 840

gagcctgcag ggaacagaac agtgaacagc gaggactctg atgaacaaga ccctcaggag 900

gtgacataca cacagttgaa tcactgcgtt ttcacacaga gaaaaatcac tcgcccttct 960

cagaggccca agacaccccc aacagatatc atcgtgtaca cggaacttcc aaatgctgag 1020

tccaga 1026

<210> 31

<211> 304

<212> PRT

<213> Chile person

<400> 31

Met Ser Leu Met Val Ile Ser Met Ala Cys Val Gly Phe Phe Trp Leu

1 5 10 15

Gln Gly Ala Trp Pro His Glu Gly Phe Arg Arg Lys Pro Ser Leu Leu

20 25 30

Ala His Pro Gly Arg Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln

35 40 45

Cys Trp Ser Asp Val Met Phe Glu His Phe Leu Leu His Arg Glu Gly

50 55 60

Thr Phe Asn Asp Thr Leu Arg Leu Ile Gly Glu His Ile Asp Gly Val

65 70 75 80

Ser Lys Ala Asn Phe Ser Ile Gly Arg Met Arg Gln Asp Leu Ala Gly

85 90 95

Thr Tyr Arg Cys Tyr Gly Ser Val Pro His Ser Pro Tyr Gln Phe Ser

100 105 110

Ala Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys

115 120 125

Pro Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser

130 135 140

Val Thr Leu Ser Cys Ser Ser Trp Ser Ser Tyr Asp Met Tyr His Leu

145 150 155 160

Ser Thr Glu Gly Glu Ala His Glu Arg Arg Phe Ser Ala Gly Pro Lys

165 170 175

Val Asn Gly Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr Gln

180 185 190

Gly Gly Thr Tyr Arg Cys Phe Gly Ser Phe His Asp Ser Pro Tyr Glu

195 200 205

Trp Ser Lys Ser Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro

210 215 220

Ser Asn Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn

225 230 235 240

Pro Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Lys Leu Pro

245 250 255

Phe Thr Ile Leu Leu Phe Phe Leu Leu His Arg Trp Cys Ser Asp Lys

260 265 270

Lys Asn Ala Ser Val Met Asp Gln Gly Pro Ala Gly Asn Arg Thr Val

275 280 285

Asn Arg Glu Asp Ser Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala

290 295 300

<210> 32

<211> 915

<212> DNA

<213> Chile person

<400> 32

atgtcgctca tggtcatcag catggcatgt gttgggttct tctggctgca gggggcctgg 60

ccacatgagg gattccgcag aaaaccttcc ctcctggccc acccaggtcg cctggtgaaa 120

tcagaagaga cagtcatcct gcaatgttgg tcagatgtca tgtttgagca cttccttctg 180

cacagagagg ggacgtttaa cgacactttg cgcctcattg gagagcacat tgatggggtc 240

tccaaggcca acttctccat cggtcgcatg aggcaagacc tggcagggac ctacagatgc 300

tacggttctg ttcctcactc cccctatcag ttttcagctc ccagtgaccc tctggacatc 360

gtgatcacag gtctatatga gaaaccttct ctctcagccc agccgggccc cacggttctg 420

gcaggagaga gcgtgacctt gtcctgcagc tcctggagct cctatgacat gtaccatcta 480

tccacggagg gggaggccca tgaacgtagg ttctctgcag ggcccaaggt caacggaaca 540

ttccaggccg actttcctct gggccctgcc acccaaggag gaacctacag atgcttcggc 600

tctttccatg actctcccta cgagtggtca aagtcaagtg acccactgct tgtttctgtc 660

acaggaaacc cttcaaatag ttggccttca cccactgaac caagctccaa aaccggtaac 720

cccagacacc tacacgttct gattgggacc tcagtggtca aactcccttt caccatcctc 780

ctcttctttc tccttcatcg ctggtgctcc gacaaaaaaa atgcatctgt aatggaccaa 840

gggcctgcgg ggaacagaac agtgaacagg gaggattctg atgaacagga ccatcaggag 900

gtgtcatacg cataa 915

<210> 33

<211> 354

<212> PRT

<213> Chile person

<400> 33

His Val Gly Gly Gln Asp Lys Pro Phe Cys Ser Ala Trp Pro Ser Ala

1 5 10 15

Val Val Pro Gln Gly Gly His Ala Thr Leu Arg Cys His Cys Arg Arg

20 25 30

Gly Phe Asn Ile Phe Thr Leu Tyr Lys Lys Asp Gly Val Pro Val Pro

35 40 45

Glu Leu Tyr Asn Arg Ile Phe Trp Asn Ser Phe Leu Ile Ser Pro Val

50 55 60

Thr Pro Ala His Ala Gly Thr Tyr Arg Cys Arg Gly Phe His Pro His

65 70 75 80

Ser Pro Thr Glu Trp Ser Ala Pro Ser Asn Pro Leu Val Ile Met Val

85 90 95

Thr Gly Leu Tyr Glu Lys Pro Ser Leu Thr Ala Arg Pro Gly Pro Thr

100 105 110

Val Arg Ala Gly Glu Asn Val Thr Leu Ser Cys Ser Ser Gln Ser Ser

115 120 125

Phe Asp Ile Tyr His Leu Ser Arg Glu Gly Glu Ala His Glu Leu Arg

130 135 140

Leu Pro Ala Val Pro Ser Ile Asn Gly Thr Phe Gln Ala Asp Phe Pro

145 150 155 160

Leu Gly Pro Ala Thr His Gly Glu Thr Tyr Arg Cys Phe Gly Ser Phe

165 170 175

His Gly Ser Pro Tyr Glu Trp Ser Asp Pro Ser Asp Pro Leu Pro Val

180 185 190

Ser Val Thr Gly Asn Pro Ser Ser Ser Trp Pro Ser Pro Thr Glu Pro

195 200 205

Ser Phe Lys Thr Gly Ile Ala Arg His Leu His Ala Val Ile Arg Tyr

210 215 220

Ser Val Ala Ile Ile Leu Phe Thr Ile Leu Pro Phe Phe Leu Leu His

225 230 235 240

Arg Trp Cys Ser Lys Lys Lys Asn Ala Ala Val Met Asn Gln Glu Pro

245 250 255

Ala Gly His Arg Thr Val Asn Arg Glu Asp Ser Asp Glu Gln Asp Pro

260 265 270

Gln Glu Val Thr Tyr Ala Gln Leu Asp His Cys Ile Phe Thr Gln Arg

275 280 285

Lys Ile Thr Gly Pro Ser Gln Arg Ser Lys Arg Pro Ser Thr Asp Thr

290 295 300

Ser Val Cys Ile Glu Leu Pro Asn Ala Glu Pro Arg Ala Leu Ser Pro

305 310 315 320

Ala His Glu His His Ser Gln Ala Leu Met Gly Ser Ser Arg Glu Thr

325 330 335

Thr Ala Leu Ser Gln Thr Gln Leu Ala Ser Ser Asn Val Pro Ala Ala

340 345 350

Gly Ile

<210> 34

<211> 1251

<212> DNA

<213> Chile person

<400> 34

atgtcccctt cacatgttgt ggtcaatgtg tcaactgcac gatccgggcc cctcaccaca 60

tcctctgcac cggtcagtcg agccgagtca ctgcgtcctg gcagcagaag ctgcaccatg 120

tccatgtcac ccacggtcat catcctggca tgtcttgggt tcttcttgga ccagagtgtg 180

tgggcacacg tgggtggtca ggacaagccc ttctgctctg cctggcccag cgctgtggtg 240

cctcaaggag gacacgtgac tcttcggtgt cactatcgtc gtgggtttaa catcttcacg 300

ctgtacaaga aagatggggt ccctgtccct gagctctaca acagaatatt ctggaacagt 360

ttcctcatta gccctgtgac cccagcacac gcagggacct acagatgtcg aggttttcac 420

ccgcactccc ccactgagtg gtcggcaccc agcaaccccc tggtgatcat ggtcacaggt 480

ctatatgaga aaccttcgct tacagcccgg ccgggcccca cggttcgcgc aggagagaac 540

gtgaccttgt cctgcagctc ccagagctcc tttgacatct accatctatc cagggagggg 600

gaagcccatg aacttaggct ccctgcagtg cccagcatca atggaacatt ccaggccgac 660

ttccctctgg gtcctgccac ccacggagag acctacagat gcttcggctc tttccatgga 720

tctccctacg agtggtcaga cccgagtgac ccactgcctg tttctgtcac aggaaaccct 780

tctagtagtt ggccttcacc cactgaacca agcttcaaaa ctggtatcgc cagacacctg 840

catgctgtga ttaggtactc agtggccatc atcctcttta ccatccttcc cttctttctc 900

cttcatcgct ggtgctccaa aaaaaaagat gctgctgtaa tgaaccaaga gcctgcggga 960

cacagaacag tgaacaggga ggactctgat gaacaagacc ctcaggaggt gacatacgca 1020

cagttggatc actgcatttt cacacagaga aaaatcactg gcccttctca gaggagcaag 1080

agaccctcaa cagataccag cgtgtgtata gaacttccaa atgctgagcc cagagcgttg 1140

tctcctgccc atgagcacca cagtcaggcc ttgatgggat cttctaggga gacaacagcc 1200

ctgtctcaaa cccagcttgc cagctctaat gtaccagcag ctggaatctg a 1251

<210> 35

<211> 218

<212> PRT

<213> Chile person

<400> 35

Gln Glu Gly Val His Arg Lys Pro Ser Phe Leu Ala Leu Pro Gly His

1 5 10 15

Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val

20 25 30

Met Phe Glu His Phe Leu Leu His Arg Glu Gly Lys Phe Asn Asn Thr

35 40 45

Leu His Leu Ile Gly Glu His His Asp Gly Val Ser Lys Ala Asn Phe

50 55 60

Ser Ile Gly Pro Met Met Pro Val Leu Ala Gly Thr Tyr Arg Cys Tyr

65 70 75 80

Ser Ser Val Pro His Ser Pro Tyr Gln Leu Ser Ala Pro Ser Asp Pro

85 90 95

Leu Asp Met Val Ile Ile Gly Leu Tyr Glu Lys Pro Ser Leu Ser Ala

100 105 110

Gln Pro Gly Pro Thr Val Gln Ala Gly Glu Asn Val Ser Leu Ser Cys

115 120 125

Ser Ser Ile Tyr Pro Gly Arg Gly Arg Pro Met Asn Val Gly Ser Leu

130 135 140

Gln Cys Ala Ala Ser Thr Glu His Ser Arg Pro Thr Phe Leu Trp Ala

145 150 155 160

Leu Pro Pro Thr Glu Gly Pro Thr Asp Ala Ser Ala Leu Ser Val Thr

165 170 175

Leu Pro Thr Ser Gly Gln Thr Arg Val Ile His Cys Leu Phe Pro Ser

180 185 190

Gln Glu Thr Leu Gln Ile Val Gly Leu His Pro Leu Asn Gln Ala Pro

195 200 205

Lys Pro Val Thr Pro Asp Thr Tyr Met Phe

210 215

<210> 36

<211> 893

<212> DNA

<213> Chile person

<400> 36

atgtcgctca tggtcatcat catggcgtgt gttgggttct tcttgctgca gggggcctgg 60

ccacaggagg gagtccacag aaaaccttcc ttcctggccc tcccaggtca cctggtgaaa 120

tcagaagaga cagtcatcct gcaatgttgg tcggatgtca tgtttgagca cttccttctg 180

cacagagagg ggaagtttaa caacactttg cacctcattg gagagcacca tgatggggtt 240

tccaaggcca acttctccat tggtcccatg atgcctgtcc ttgcaggaac ctacagatgc 300

tacggttctg ttcctcactc cccctatcag ttgtcagctc ccagtgaccc tctggacatg 360

gtgatcatag gtctatatga gaaaccttct ctctcagccc agccgggccc cacggttcag 420

gcaggagaga atgtgacctt gtcctgcagc tccatctatc cagggaaggg gaggcccatg 480

aacgtaggct ccctgcagtg cgcagcatca acggaacatt ccaggccgac tttcctctgg 540

gccctgccac ccacggaggg acctacagat gcttcggctc tttccgtgac gctccctacg 600

agtggtcaaa ctcgagtgat ccactgcttg tttccgtcac aggaaaccct tcaaatagtt 660

ggccttcacc cactgaacca agctccaaaa ccggtaaccc cagacaccta catgttctga 720

ttgggacctc agtggtcaaa atccctttca ccatcctcct cttctttctc cttcatcgct 780

ggtgctccga caaaaaaaat gctgctgtaa tggaccaaga gcctgcaggg aacagaacag 840

tgaacagcga ggattctgat gaacaagacc atcaggaggt gtcatacgca taa 893

<210> 37

<211> 283

<212> PRT

<213> Chile person

<400> 37

His Glu Gly Phe Arg Arg Lys Pro Ser Leu Leu Ala His Pro Gly Pro

1 5 10 15

Leu Val Lys Ser Glu Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val

20 25 30

Met Phe Glu His Phe Leu Leu His Arg Glu Gly Thr Phe Asn His Thr

35 40 45

Leu Arg Leu Ile Gly Glu His Ile Asp Gly Val Ser Lys Gly Asn Phe

50 55 60

Ser Ile Gly Arg Met Thr Gln Asp Leu Ala Gly Thr Tyr Arg Cys Tyr

65 70 75 80

Gly Ser Val Thr His Ser Pro Tyr Gln Leu Ser Ala Pro Ser Asp Pro

85 90 95

Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys Pro Ser Leu Ser Ala

100 105 110

Gln Pro Gly Pro Thr Val Leu Ala Gly Glu Ser Val Thr Leu Ser Cys

115 120 125

Ser Ser Arg Ser Ser Tyr Asp Met Tyr His Leu Ser Arg Glu Gly Glu

130 135 140

Ala His Glu Arg Arg Leu Pro Ala Gly Thr Lys Val Asn Gly Thr Phe

145 150 155 160

Gln Ala Asp Phe Pro Leu Asp Pro Ala Thr His Gly Gly Thr Tyr Arg

165 170 175

Cys Phe Gly Ser Phe Arg Asp Ser Pro Tyr Glu Trp Ser Lys Ser Ser

180 185 190

Asp Pro Leu Leu Val Ser Val Thr Gly Asn Thr Ser Asn Ser Trp Pro

195 200 205

Ser Pro Thr Glu Pro Ser Ser Lys Thr Gly Asn Pro Arg His Leu His

210 215 220

Val Leu Ile Gly Thr Ser Val Val Lys Leu Pro Phe Thr Ile Leu Leu

225 230 235 240

Phe Phe Leu Leu His Arg Trp Cys Ser Asn Lys Lys Asn Ala Ser Val

245 250 255

Met Asp Gln Gly Pro Ala Gly Asn Arg Thr Val Asn Arg Glu Asp Ser

260 265 270

Asp Glu Gln Asp His Gln Glu Val Ser Tyr Ala

275 280

<210> 38

<211> 915

<212> DNA

<213> Chile person

<400> 38

atgtcgctca tggtcatcag catggcgtgt gttgcgttct tcttgctgca gggggcctgg 60

ccacatgagg gattccgcag aaaaccttcc ctcctggccc acccaggtcc cctggtgaaa 120

tcagaagaga cagtcatcct gcaatgttgg tcagatgtca tgtttgagca cttccttctg 180

cacagagagg ggacgtttaa ccacactttg cgcctcattg gagagcacat tgatggggtc 240

tccaagggca acttctccat cggtcgcatg acacaagacc tggcagggac ctacagatgc 300

tacggttctg ttactcactc cccctatcag ttgtcagcgc ccagtgaccc tctggacatc 360

gtgatcacag gtctatatga gaaaccttct ctctcagccc agccgggccc cacggttctg 420

gcaggagaga gcgtgacctt gtcctgcagc tcccggagct cctatgacat gtaccatcta 480

tccagggaag gggaggccca tgaacgtagg ctccctgcag ggcccaaggt caacagaaca 540

ttccaggccg actttcctct ggaccctgcc acccacggag ggacctacag atgcttcggc 600

tctttccgtg actctccata cgagtggtca aagtcaagtg acccactgct tgtttctgtc 660

acaggaaact cttcaaatag ttggccttca cccactgaac caagctccga aaccggtaac 720

cccagacacc tacacgttct gattgggacc tcagtggtca aactcccttt caccatcctc 780

ctcttctttc tccttcatcg ctggtgctcc aacaaaaaaa atgcatctgt aatggaccaa 840

gggcctgcgg ggaacagaac agtgaacagg gaggattctg atgaacagga ccatcaggag 900

gtgtcatacg cataa 915

<210> 39

<211> 361

<212> PRT

<213> Chile person

<400> 39

His Met Gly Gly Gln Asp Lys Pro Phe Leu Ser Ala Trp Pro Ser Ala

1 5 10 15

Val Val Pro Arg Gly Gly His Val Thr Leu Arg Cys His Tyr Arg His

20 25 30

Arg Phe Asn Asn Phe Met Leu Tyr Lys Glu Asp Arg Ile His Val Pro

35 40 45

Ile Phe His Gly Arg Ile Phe Gln Glu Gly Phe Asn Met Ser Pro Val

50 55 60

Thr Thr Ala His Ala Gly Asn Tyr Thr Cys Arg Gly Ser His Pro His

65 70 75 80

Ser Pro Thr Gly Trp Ser Ala Pro Ser Asn Pro Met Val Ile Met Val

85 90 95

Thr Gly Asn His Arg Lys Pro Ser Leu Leu Ala His Pro Gly Pro Leu

100 105 110

Val Lys Ser Gly Glu Arg Val Ile Leu Gln Cys Trp Ser Asp Ile Met

115 120 125

Phe Glu His Phe Phe Leu His Lys Glu Gly Ile Ser Lys Asp Pro Ser

130 135 140

Arg Leu Val Gly Gln Ile His Asp Gly Val Ser Lys Ala Asn Phe Ser

145 150 155 160

Ile Gly Ser Met Met Arg Ala Leu Ala Gly Thr Tyr Arg Cys Tyr Gly

165 170 175

Ser Val Thr His Thr Pro Tyr Gln Leu Ser Ala Pro Ser Asp Pro Leu

180 185 190

Asp Ile Val Val Thr Gly Leu Tyr Glu Lys Pro Ser Leu Ser Ala Gln

195 200 205

Pro Gly Pro Lys Val Gln Ala Gly Glu Ser Val Thr Leu Ser Cys Ser

210 215 220

Ser Arg Ser Ser Tyr Asp Met Tyr His Leu Ser Arg Glu Gly Gly Ala

225 230 235 240

His Glu Arg Arg Leu Pro Ala Val Arg Lys Val Asn Arg Thr Phe Gln

245 250 255

Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly Gly Thr Tyr Arg Cys

260 265 270

Phe Gly Ser Phe Arg His Ser Pro Tyr Glu Trp Ser Asp Pro Ser Asp

275 280 285

Pro Leu Leu Val Ser Val Thr Gly Asn Pro Ser Ser Ser Trp Pro Ser

290 295 300

Pro Thr Glu Pro Ser Ser Lys Ser Gly Asn Leu Arg His Leu His Ile

305 310 315 320

Leu Ile Gly Thr Ser Val Val Lys Ile Pro Phe Thr Ile Leu Leu Phe

325 330 335

Phe Leu Leu His Arg Trp Cys Ser Asn Lys Lys Lys Cys Cys Cys Asn

340 345 350

Gly Pro Arg Ala Cys Arg Glu Gln Lys

355 360

<210> 40

<211> 1149

<212> DNA

<213> Chile person

<400> 40

atgttgctca tggtcgtcag catggcgtgt gttgggttgt tcttggtcca gagggccggt 60

ccacacatgg gtggtcagga caagcccttc ctgtctgcct ggcccagcgc tgtggtgcct 120

cgcggaggac acgtgactct tcggtgtcac tatcgtcata ggtttaacaa tttcatgcta 180

tacaaagaag acagaatcca cgttcccatc ttccatggca gaatattcca ggagggcttc 240

aacatgagcc ctgtgaccac agcacatgca gggaactaca catgtcgggg ttcacaccca 300

cactccccca ctgggtggtc ggcacccagc aaccccatgg tgatcatggt cacaggaaac 360

cacagaaaac cttccctcct ggcccaccca ggtcccctgg tgaaatcagg agagagagtc 420

atcctgcaat gttggtcaga tatcatgttt gagcacttct ttctgcacaa agagtggatc 480

tctaaggacc cctcacgcct cgttggacag atccatgatg gggtctccaa ggccaatttc 540

tccatcggtt ccatgatgcg tgcccttgca gggacctaca gatgctacgg ttctgttact 600

cacaccccct atcagttgtc agctcccagt gatcccctgg acatcgtggt cacaggtcta 660

tatgagaaac cttctctctc agcccagccg ggccccaagg ttcaggcagg agagagcgtg 720

accttgtcct gtagctcccg gagctcctat gacatgtacc atctatccag ggagggggga 780

gcccatgaac gtaggctccc tgcagtgcgc aaggtcaaca gaacattcca ggcagatttc 840

cctctgggcc ctgccaccca cggagggacc tacagatgct tcggctcttt ccgtcactct 900

ccctacgagt ggtcagaccc gagtgaccca ctgcttgttt ctgtcacagg aaacccttca 960

agtagttggc cttcacccac agaaccaagc tccaaatctg gtaacctcag acacctgcac 1020

attctgattg ggacctcagt ggtcaaaatc cctttcacca tcctcctctt ctttctcctt 1080

catcgctggt gctccaacaa aaaaaaatgc tgctgtaatg gaccaagagc ctgcagggaa 1140

cagaagtga 1149

<210> 41

<211> 231

<212> PRT

<213> Chile person

<400> 41

Met Ser Lys Gln Arg Gly Thr Phe Ser Glu Val Ser Leu Ala Gln Asp

1 5 10 15

Pro Lys Arg Gln Gln Arg Lys Pro Lys Gly Asn Lys Ser Ser Ile Ser

20 25 30

Gly Thr Glu Gln Glu Ile Phe Gln Val Glu Leu Asn Leu Gln Asn Pro

35 40 45

Ser Leu Asn His Gln Gly Ile Asp Lys Ile Tyr Asp Cys Gln Gly Leu

50 55 60

Leu Pro Pro Pro Glu Lys Leu Thr Ala Glu Val Leu Gly Ile Ile Cys

65 70 75 80

Ile Val Leu Met Ala Thr Val Leu Lys Thr Ile Val Leu Ile Pro Phe

85 90 95

Leu Glu Gln Asn Asn Ser Ser Pro Asn Thr Arg Thr Gln Lys Ala Arg

100 105 110

His Cys Gly His Cys Pro Glu Glu Trp Ile Thr Tyr Ser Asn Ser Cys

115 120 125

Tyr Tyr Ile Gly Lys Glu Arg Arg Thr Trp Glu Glu Ser Leu Leu Ala

130 135 140

Cys Thr Ser Lys Asn Ser Ser Leu Leu Ser Ile Asp Asn Glu Glu Glu

145 150 155 160

Met Lys Phe Leu Ala Ser Ile Leu Pro Ser Ser Trp Ile Gly Val Phe

165 170 175

Arg Asn Ser Ser His His Pro Trp Val Thr Ile Asn Gly Leu Ala Phe

180 185 190

Lys His Lys Ile Lys Asp Ser Asp Asn Ala Glu Leu Asn Cys Ala Val

195 200 205

Leu Gln Val Asn Arg Leu Lys Ser Ala Gln Cys Gly Ser Ser Met Ile

210 215 220

Tyr His Cys Lys His Lys Leu

225 230

<210> 42

<211> 696

<212> DNA

<213> Chile person

<400> 42

atgaataaac aaagaggaac cttctcagaa gtgagtctgg cccaggaccc aaagcggcag 60

caaaggaaac ctaaaggcaa taaaagctcc atttcaggaa ccgaacagga aatattccaa 120

gtagaattaa atcttcaaaa tccttccctg aatcatcaag ggattgataa aatatatgac 180

tgccaaggtt tactgccacc tccagagaag ctcactgccg aggtcctagg aatcatttgc 240

attgtcctga tggccactgt gttaaaaaca atagttctta ttcctttcct ggagcagaac 300

aatttttccc cgaatacaag aacgcagaaa gcacgtcatt gtggccattg tcctgaggag 360

tggattacat attccaacag ttgttattac attggtaagg aaagaagaac ttgggaagag 420

agtttgctgg cctgtacttc gaagaactcc agtctgcttt ctatagataa tgaagaagaa 480

atgaaatttc tggccagcat tttaccttcc tcatggattg gtgtgtttcg taacagcagt 540

catcatccat gggtgacaat aaatggtttg gctttcaaac ataagataaa agactcagat 600

aatgctgaac ttaactgtgc agtgctacaa gtaaatcgac ttaaatcagc ccagtgtgga 660

tcttcaatga tatatcattg taagcataag ctttag 696

<210> 43

<211> 354

<212> PRT

<213> Chile person

<400> 43

Gln Asp Glu Val Thr Asp Asp Tyr Ile Gly Asp Asn Thr Thr Val Asp

1 5 10 15

Tyr Thr Leu Phe Glu Ser Leu Cys Ser Lys Lys Asp Val Arg Asn Phe

20 25 30

Lys Ala Trp Phe Leu Pro Ile Met Tyr Ser Ile Ile Cys Phe Val Gly

35 40 45

Leu Leu Gly Asn Gly Leu Val Val Leu Thr Tyr Ile Tyr Phe Lys Arg

50 55 60

Leu Lys Thr Met Thr Asp Thr Tyr Leu Leu Asn Leu Ala Val Ala Asp

65 70 75 80

Ile Leu Phe Leu Leu Thr Leu Pro Phe Trp Ala Tyr Ser Ala Ala Lys

85 90 95

Ser Trp Val Phe Gly Val His Phe Cys Lys Leu Ile Phe Ala Ile Tyr

100 105 110

Lys Met Ser Phe Phe Ser Gly Met Leu Leu Leu Leu Cys Ile Ser Ile

115 120 125

Asp Arg Tyr Val Ala Ile Val Gln Ala Val Ser Ala His Arg His Arg

130 135 140

Ala Arg Val Leu Leu Ile Ser Lys Leu Ser Cys Val Gly Ile Trp Ile

145 150 155 160

Leu Ala Thr Val Leu Ser Ile Pro Glu Leu Leu Tyr Ser Asp Leu Gln

165 170 175

Arg Ser Ser Ser Glu Gln Ala Met Arg Cys Ser Leu Ile Thr Glu His

180 185 190

Val Glu Ala Phe Ile Thr Ile Gln Val Ala Gln Met Val Ile Gly Phe

195 200 205

Leu Val Pro Leu Leu Ala Met Ser Phe Cys Tyr Leu Val Ile Ile Arg

210 215 220

Thr Leu Leu Gln Ala Arg Asn Phe Glu Arg Asn Lys Ala Ile Lys Val

225 230 235 240

Ile Ile Ala Val Val Val Val Phe Ile Val Phe Gln Leu Pro Tyr Asn

245 250 255

Gly Val Val Leu Ala Gln Thr Val Ala Asn Phe Asn Ile Thr Ser Ser

260 265 270

Thr Cys Glu Leu Ser Lys Gln Leu Asn Ile Ala Tyr Asp Val Thr Tyr

275 280 285

Ser Leu Ala Cys Val Arg Cys Cys Val Asn Pro Phe Leu Tyr Ala Phe

290 295 300

Ile Gly Val Lys Phe Arg Asn Asp Leu Phe Lys Leu Phe Lys Asp Leu

305 310 315 320

Gly Cys Leu Ser Gln Glu Gln Leu Arg Gln Trp Ser Ser Cys Arg His

325 330 335

Ile Arg Arg Ser Ser Met Ser Val Glu Ala Glu Thr Thr Thr Thr Phe

340 345 350

Ser Pro

<210> 44

<211> 1137

<212> DNA

<213> Chile person

<400> 44

atggacctgg ggaaaccaat gaaaagcgtg ctggtggtgg ctctccttgt cattttccag 60

gtatgcctgt gtcaagatga ggtcacggac gattacatcg gagacaacac cacagtggac 120

tacactttgt tcgagtcttt gtgctccaag aaggacgtgc ggaactttaa agcctggttc 180

ctccctatca tgtactccat catttgtttc gtgggcctac tgggcaatgg gctggtcgtg 240

ttgacctata tctatttcaa gaggctcaag accatgaccg atacctacct gctcaacctg 300

gcggtggcag acatcctctt cctcctgacc cttcccttct gggcctacag cgcggccaag 360

tcctgggtct tcggtgtcca cttttgcaag ctcatctttg ccatctacaa gatgagcttc 420

ttcagtggca tgctcctact tctttgcatc agcattgacc gctacgtggc catcgtccag 480

gctgtctcag ctcaccgcca ccgtgcccgc gtccttctca tcagcaagct gtcctgtgtg 540

ggcatctgga tactagccac agtgctctcc atcccagagc tcctgtacag tgacctccag 600

aggagcagca gtgagcaagc gatgcgatgc tctctcatca cagagcatgt ggaggccttt 660

atcaccatcc aggtggccca gatggtgatc ggctttctgg tccccctgct ggccatgagc 720

ttctgttacc ttgtcatcat ccgcaccctg ctccaggcac gcaactttga gcgcaacaag 780

gccatcaagg tgatcatcgc tgtggtcgtg gtcttcatag tcttccagct gccctacaat 840

ggggtggtcc tggcccagac ggtggccaac ttcaacatca ccagtagcac ctgtgagctc 900

agtaagcaac tcaacatcgc ctacgacgtc acctacagcc tggcctgcgt ccgctgctgc 960

gtcaaccctt tcttgtacgc cttcatcggc gtcaagttcc gcaacgatct cttcaagctc 1020

ttcaaggacc tgggctgcct cagccaggag cagctccggc agtggtcttc ctgtcggcac 1080

atccggcgct cctccatgag tgtggaggcc gagaccacca ccaccttctc cccatag 1137

<210> 45

<211> 368

<212> PRT

<213> Chile person

<400> 45

Met Val Leu Glu Val Ser Asp His Gln Val Leu Asn Asp Ala Glu Val

1 5 10 15

Ala Ala Leu Leu Glu Asn Phe Ser Ser Ser Tyr Asp Tyr Gly Glu Asn

20 25 30

Glu Ser Asp Ser Cys Cys Thr Ser Pro Pro Cys Pro Gln Asp Phe Ser

35 40 45

Leu Asn Phe Asp Arg Ala Phe Leu Pro Ala Leu Tyr Ser Leu Leu Phe

50 55 60

Leu Leu Gly Leu Leu Gly Asn Gly Ala Val Ala Ala Val Leu Leu Ser

65 70 75 80

Arg Arg Thr Ala Leu Ser Ser Thr Asp Thr Phe Leu Leu His Leu Ala

85 90 95

Val Ala Asp Thr Leu Leu Val Leu Thr Leu Pro Leu Trp Ala Val Asp

100 105 110

Ala Ala Val Gln Trp Val Phe Gly Ser Gly Leu Cys Lys Val Ala Gly

115 120 125

Ala Leu Phe Asn Ile Asn Phe Tyr Ala Gly Ala Leu Leu Leu Ala Cys

130 135 140

Ile Ser Phe Asp Arg Tyr Leu Asn Ile Val His Ala Thr Gln Leu Tyr

145 150 155 160

Arg Arg Gly Pro Pro Ala Arg Val Thr Leu Thr Cys Leu Ala Val Trp

165 170 175

Gly Leu Cys Leu Leu Phe Ala Leu Pro Asp Phe Ile Phe Leu Ser Ala

180 185 190

His His Asp Glu Arg Leu Asn Ala Thr His Cys Gln Tyr Asn Phe Pro

195 200 205

Gln Val Gly Arg Thr Ala Leu Arg Val Leu Gln Leu Val Ala Gly Phe

210 215 220

Leu Leu Pro Leu Leu Val Met Ala Tyr Cys Tyr Ala His Ile Leu Ala

225 230 235 240

Val Leu Leu Val Ser Arg Gly Gln Arg Arg Leu Arg Ala Met Arg Leu

245 250 255

Val Val Val Val Val Val Ala Phe Ala Leu Cys Trp Thr Pro Tyr His

260 265 270

Leu Val Val Leu Val Asp Ile Leu Met Asp Leu Gly Ala Leu Ala Arg

275 280 285

Asn Cys Gly Arg Glu Ser Arg Val Asp Val Ala Lys Ser Val Thr Ser

290 295 300

Gly Leu Gly Tyr Met His Cys Cys Leu Asn Pro Leu Leu Tyr Ala Phe

305 310 315 320

Val Gly Val Lys Phe Arg Glu Arg Met Trp Met Leu Leu Leu Arg Leu

325 330 335

Gly Cys Pro Asn Gln Arg Gly Leu Gln Arg Gln Pro Ser Ser Ser Arg

340 345 350

Arg Asp Ser Ser Trp Ser Glu Thr Ser Glu Ala Ser Tyr Ser Gly Leu

355 360 365

<210> 46

<211> 1248

<212> DNA

<213> Chile person

<400> 46

atggagttga ggaagtacgg ccctggaaga ctggcgggga cagttatagg aggagctgct 60

cagagtaaat cacagactaa atcagactca atcacaaaag agttcctgcc aggcctttac 120

acagcccctt cctccccgtt cccgccctca caggtgagtg accaccaagt gctaaatgac 180

gccgaggttg ccgccctcct ggagaacttc agctcttcct atgactatgg agaaaacgag 240

agtgactcgt gctgtacctc cccgccctgc ccacaggact tcagcctgaa cttcgaccgg 300

gccttcctgc cagccctcta cagcctcctc tttctgctgg ggctgctggg caacggcgcg 360

gtggcagccg tgctgctgag ccggcggaca gccctgagca gcaccgacac cttcctgctc 420

cacctagctg tagcagacac gctgctggtg ctgacactgc cgctctgggc agtggacgct 480

gccgtccagt gggtctttgg ctctggcctc tgcaaagtgg caggtgccct cttcaacatc 540

aacttctacg caggagccct cctgctggcc tgcatcagct ttgaccgcta cctgaacata 600

gttcatgcca cccagctcta ccgccggggg cccccggccc gcgtgaccct cacctgcctg 660

gctgtctggg ggctctgcct gcttttcgcc ctcccagact tcatcttcct gtcggcccac 720

cacgacgagc gcctcaacgc cacccactgc caatacaact tcccacaggt gggccgcacg 780

gctctgcggg tgctgcagct ggtggctggc tttctgctgc ccctgctggt catggcctac 840

tgctatgccc acatcctggc cgtgctgctg gtttccaggg gccagcggcg cctgcgggcc 900

atgcggctgg tggtggtggt cgtggtggcc tttgccctct gctggacccc ctatcacctg 960

gtggtgctgg tggacatcct catggacctg ggcgctttgg cccgcaactg tggccgagaa 1020

agcagggtag acgtggccaa gtcggtcacc tcaggcctgg gctacatgca ctgctgcctc 1080

aacccgctgc tctatgcctt tgtaggggtc aagttccggg agcggatgtg gatgctgctc 1140

ttgcgcctgg gctgccccaa ccagagaggg ctccagaggc agccatcgtc ttcccgccgg 1200

gattcatcct ggtctgagac ctcagaggcc tcctactcgg gcttgtga 1248

<210> 47

<211> 344

<212> PRT

<213> Chile person

<400> 47

Asp Phe Leu Ala His His Gly Thr Asp Cys Trp Thr Tyr His Tyr Ser

1 5 10 15

Glu Lys Pro Met Asn Trp Gln Arg Ala Arg Arg Phe Cys Arg Asp Asn

20 25 30

Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys Ala Glu Ile Glu Tyr Leu

35 40 45

Glu Lys Thr Leu Pro Phe Ser Arg Ser Tyr Tyr Trp Ile Gly Ile Arg

50 55 60

Lys Ile Gly Gly Ile Trp Thr Trp Val Gly Thr Asn Lys Ser Leu Thr

65 70 75 80

Glu Glu Ala Glu Asn Trp Gly Asp Gly Glu Pro Asn Asn Lys Lys Asn

85 90 95

Lys Glu Asp Cys Val Glu Ile Tyr Ile Lys Arg Asn Lys Asp Ala Gly

100 105 110

Lys Trp Asn Asp Asp Ala Cys His Lys Leu Lys Ala Ala Leu Cys Tyr

115 120 125

Thr Ala Ser Cys Gln Pro Trp Ser Cys Ser Gly His Gly Glu Cys Val

130 135 140

Glu Ile Ile Asn Asn Tyr Thr Cys Asn Cys Asp Val Gly Tyr Tyr Gly

145 150 155 160

Pro Gln Cys Gln Phe Val Ile Gln Cys Glu Pro Leu Glu Ala Pro Glu

165 170 175

Leu Gly Thr Met Asp Cys Thr His Pro Leu Gly Asn Phe Ser Phe Ser

180 185 190

Ser Gln Cys Ala Phe Ser Cys Ser Glu Gly Thr Asn Leu Thr Gly Ile

195 200 205

Glu Glu Thr Thr Cys Gly Pro Phe Gly Asn Trp Ser Ser Pro Glu Pro

210 215 220

Thr Cys Gln Val Ile Gln Cys Glu Pro Leu Ser Ala Pro Asp Leu Gly

225 230 235 240

Ile Met Asn Cys Ser His Pro Leu Ala Ser Phe Ser Phe Thr Ser Ala

245 250 255

Cys Thr Phe Ile Cys Ser Glu Gly Thr Glu Leu Ile Gly Lys Lys Lys

260 265 270

Thr Ile Cys Glu Ser Ser Gly Ile Trp Ser Asn Pro Ser Pro Ile Cys

275 280 285

Gln Lys Leu Asp Lys Ser Phe Ser Met Ile Lys Glu Gly Asp Tyr Asn

290 295 300

Pro Leu Phe Ile Pro Val Ala Val Met Val Thr Ala Phe Ser Gly Leu

305 310 315 320

Ala Phe Ile Ile Trp Leu Ala Arg Arg Leu Lys Lys Gly Lys Lys Ser

325 330 335

Lys Arg Ser Met Asn Asp Pro Tyr

340

<210> 48

<211> 1158

<212> DNA

<213> Chile person

<400> 48

atgggctgca gaagaactag agaaggacca agcaaagcca tgatatttcc atggaaatgt 60

cagagcaccc agagggactt atggaacatc ttcaagttgt gggggtggac aatgctctgt 120

tgtgatttcc tggcacatca tggaaccgac tgctggactt accattattc tgaaaaaccc 180

atgaactggc aaagggctag aagattctgc cgagacaatt acacagattt agttgccata 240

caaaacaagg cggaaattga gtatctggag aagactctgc ctttcagtcg ttcttactac 300

tggataggaa tccggaagat aggaggaata tggacgtggg tgggaaccaa caaatctctt 360

actgaagaag cagagaactg gggagatggt gagcccaaca acaagaagaa caaggaggac 420

tgcgtggaga tctatatcaa gagaaacaaa gatgcaggca aatggaacga tgacgcctgc 480

cacaaactaa aggcagccct ctgttacaca gcttcttgcc agccctggtc atgcagtggc 540

catggagaat gtgtagaaat catcaataat tacacctgca actgtgatgt ggggtactat 600

gggccccagt gtcagtttgt gattcagtgt gagcctttgg aggccccaga gctgggtacc 660

atggactgta ctcacccttt gggaaacttc agcttcagct cacagtgtgc cttcagctgc 720

tctgaaggaa caaacttaac tgggattgaa gaaaccacct gtggaccatt tggaaactgg 780

tcatctccag aaccaacctg tcaagtgatt cagtgtgagc ctctatcagc accagatttg 840

gggatcatga actgtagcca tcccctggcc agcttcagct ttacctctgc atgtaccttc 900

atctgctcag aaggaactga gttaattggg aagaagaaaa ccatttgtga atcatctgga 960

atctggtcaa atcctagtcc aatatgtcaa aaattggaca aaagtttctc aatgattaag 1020

gagggtgatt ataaccccct cttcattcca gtggcagtca tggttactgc attctctggg 1080

ttggcattta tcatttggct ggcaaggaga ttaaaaaaag gcaagaaatc caagagaagt 1140

atgaatgacc catattaa 1158

<210> 49

<211> 350

<212> PRT

<213> Chile person

<220>

<221> MOD_RES

<222> (31)..(31)

<223> any amino acid

<400> 49

Met Ser Asn Ile Thr Asp Pro Gln Met Trp Asp Phe Asp Asp Leu Asn

1 5 10 15

Phe Thr Gly Met Pro Pro Ala Asp Glu Asp Tyr Ser Pro Cys Xaa Leu

20 25 30

Glu Thr Glu Thr Leu Asn Lys Tyr Val Val Ile Ile Ala Tyr Ala Leu

35 40 45

Val Phe Leu Leu Ser Leu Leu Gly Asn Ser Leu Val Met Leu Val Ile

50 55 60

Leu Tyr Ser Arg Val Gly Arg Ser Val Thr Asp Val Tyr Leu Leu Asn

65 70 75 80

Leu Ala Leu Ala Asp Leu Leu Phe Ala Leu Thr Leu Pro Ile Trp Ala

85 90 95

Ala Ser Lys Val Asn Gly Trp Ile Phe Gly Thr Phe Leu Cys Lys Val

100 105 110

Val Ser Leu Leu Lys Glu Val Asn Phe Tyr Ser Gly Ile Leu Leu Leu

115 120 125

Ala Cys Ile Ser Val Asp Arg Tyr Leu Ala Ile Val His Ala Thr Arg

130 135 140

Thr Leu Thr Gln Lys Arg His Leu Val Lys Phe Val Cys Leu Gly Cys

145 150 155 160

Trp Gly Leu Ser Met Asn Leu Ser Leu Pro Phe Phe Leu Phe Arg Gln

165 170 175

Ala Tyr His Pro Asn Asn Ser Ser Pro Val Cys Tyr Glu Val Leu Gly

180 185 190

Asn Asp Thr Ala Lys Trp Arg Met Val Leu Arg Ile Leu Pro His Thr

195 200 205

Phe Gly Phe Ile Val Pro Leu Phe Val Met Leu Phe Cys Tyr Gly Phe

210 215 220

Thr Leu Arg Thr Leu Phe Lys Ala His Met Gly Gln Lys His Arg Ala

225 230 235 240

Met Arg Val Ile Phe Ala Val Val Leu Ile Phe Leu Leu Cys Trp Leu

245 250 255

Pro Tyr Asn Leu Val Leu Leu Ala Asp Thr Leu Met Arg Thr Gln Val

260 265 270

Ile Gln Glu Ser Cys Glu Arg Arg Asn Asn Ile Gly Arg Ala Leu Asp

275 280 285

Ala Thr Glu Ile Leu Gly Phe Leu His Ser Cys Leu Asn Pro Ile Ile

290 295 300

Tyr Ala Phe Ile Gly Gln Asn Phe Arg His Gly Phe Leu Lys Ile Leu

305 310 315 320

Ala Met His Gly Leu Val Ser Lys Glu Phe Leu Ala Arg His Arg Val

325 330 335

Thr Ser Tyr Thr Ser Ser Ser Val Asn Val Ser Ser Asn Leu

340 345 350

<210> 50

<211> 1053

<212> DNA

<213> Chile person

<400> 50

atgtcaaata ttacagatcc acagatgtgg gattttgatg atctaaattt cactggcatg 60

ccacctgcag atgaagatta cagcccctgt atgctagaaa ctgagacact caacaagtat 120

gttgtgatca tcgcctatgc cctagtgttc ctgctgagcc tgctgggaaa ctccctggtg 180

atgctggtca tcttatacag cagggtcggc cgctccgtca ctgatgtcta cctgctgaac 240

ctggccttgg ccgacctact ctttgccctg accttgccca tctgggccgc ctccaaggtg 300

aatggctgga tttttggcac attcctgtgc aaggtggtct cactcctgaa ggaagtcaac 360

ttctacagtg gcatcctgct gttggcctgc atcagtgtgg accgttacct ggccattgtc 420

catgccacac gcacactgac ccagaagcgt cacttggtca agtttgtttg tcttggctgc 480

tggggactgt ctatgaatct gtccctgccc ttcttccttt tccgccaggc ttaccatcca 540

aacaattcca gtccagtttg ctatgaggtc ctgggaaatg acacagcaaa atggcggatg 600

gtgttgcgga tcctgcctca cacctttggc ttcatcgtgc cgctgtttgt catgctgttc 660

tgctatggat tcaccctgcg tacactgttt aaggcccaca tggggcagaa gcaccgagcc 720

atgagggtca tctttgctgt cgtcctcatc ttcctgcttt gctggctgcc ctacaacctg 780

gtcctgctgg cagacaccct catgaggacc caggtgatcc aggagagctg tgagcgccgc 840

aacaacatcg gccgggccct ggatgccact gagattctgg gatttctcca tagctgcctc 900

aaccccatca tctacgcctt catcggccaa aattttcgcc atggattcct caagatcctg 960

gctatgcatg gcctggtcag caaggagttc ttggcacgtc atcgtgttac ctcctacact 1020

tcttcgtctg tcaatgtctc ttccaacctc tga 1053

<210> 51

<211> 360

<212> PRT

<213> Chile person

<400> 51

Met Glu Asp Phe Asn Met Glu Ser Asp Ser Phe Glu Asp Phe Trp Lys

1 5 10 15

Gly Glu Asp Leu Ser Asn Tyr Ser Tyr Ser Ser Thr Leu Pro Pro Phe

20 25 30

Leu Leu Asp Ala Ala Pro Cys Glu Pro Glu Ser Leu Glu Ile Asn Lys

35 40 45

Tyr Phe Val Val Ile Ile Tyr Ala Leu Val Phe Leu Leu Ser Leu Leu

50 55 60

Gly Asn Ser Leu Val Met Leu Val Ile Leu Tyr Ser Arg Val Gly Arg

65 70 75 80

Ser Val Thr Asp Val Tyr Leu Leu Asn Leu Ala Leu Ala Asp Leu Leu

85 90 95

Phe Ala Leu Thr Leu Pro Ile Trp Ala Ala Ser Lys Val Asn Gly Trp

100 105 110

Ile Phe Gly Thr Phe Leu Cys Lys Val Val Ser Leu Leu Lys Glu Val

115 120 125

Asn Phe Tyr Ser Gly Ile Leu Leu Leu Ala Cys Ile Ser Val Asp Arg

130 135 140

Tyr Leu Ala Ile Val His Ala Thr Arg Thr Leu Thr Gln Lys Arg Tyr

145 150 155 160

Leu Val Lys Phe Ile Cys Leu Ser Ile Trp Gly Leu Ser Leu Leu Leu

165 170 175

Ala Leu Pro Val Leu Leu Phe Arg Arg Thr Val Tyr Ser Ser Asn Val

180 185 190

Ser Pro Ala Cys Tyr Glu Asp Met Gly Asn Asn Thr Ala Asn Trp Arg

195 200 205

Met Leu Leu Arg Ile Leu Pro Gln Ser Phe Gly Phe Ile Val Pro Leu

210 215 220

Leu Ile Met Leu Phe Cys Tyr Gly Phe Thr Leu Arg Thr Leu Phe Lys

225 230 235 240

Ala His Met Gly Gln Lys His Arg Ala Met Arg Val Ile Phe Ala Val

245 250 255

Val Leu Ile Phe Leu Leu Cys Trp Leu Pro Tyr Asn Leu Val Leu Leu

260 265 270

Ala Asp Thr Leu Met Arg Thr Gln Val Ile Gln Glu Thr Cys Glu Arg

275 280 285

Arg Asn His Ile Asp Arg Ala Leu Asp Ala Thr Glu Ile Leu Gly Ile

290 295 300

Leu His Ser Cys Leu Asn Pro Leu Ile Tyr Ala Phe Ile Gly Gln Lys

305 310 315 320

Phe Arg His Gly Leu Leu Lys Ile Leu Ala Ile His Gly Leu Ile Ser

325 330 335

Lys Asp Ser Leu Pro Lys Asp Ser Arg Pro Ser Phe Val Gly Ser Ser

340 345 350

Ser Gly His Thr Ser Thr Thr Leu

355 360

<210> 52

<211> 1083

<212> DNA

<213> Chile person

<400> 52

atggaagatt ttaacatgga gagtgacagc tttgaagatt tctggaaagg tgaagatctt 60

agtaattaca gttacagctc taccctgccc ccttttctac tagatgccgc cccatgtgaa 120

ccagaatccc tggaaatcaa caagtatttt gtggtcatta tctatgccct ggtattcctg 180

ctgagcctgc tgggaaactc cctcgtgatg ctggtcatct tatacagcag ggtcggccgc 240

tccgtcactg atgtctacct gctgaaccta gccttggccg acctactctt tgccctgacc 300

ttgcccatct gggccgcctc caaggtgaat ggctggattt ttggcacatt cctgtgcaag 360

gtggtctcac tcctgaagga agtcaacttc tatagtggca tcctgctact ggcctgcatc 420

agtgtggacc gttacctggc cattgtccat gccacacgca cactgaccca gaagcgctac 480

ttggtcaaat tcatatgtct cagcatctgg ggtctgtcct tgctcctggc cctgcctgtc 540

ttacttttcc gaaggaccgt ctactcatcc aatgttagcc cagcctgcta tgaggacatg 600

ggcaacaata cagcaaactg gcggatgctg ttacggatcc tgccccagtc ctttggcttc 660

atcgtgccac tgctgatcat gctgttctgc tacggattca ccctgcgtac gctgtttaag 720

gcccacatgg ggcagaagca ccgggccatg cgggtcatct ttgctgtcgt cctcatcttc 780

ctgctctgct ggctgcccta caacctggtc ctgctggcag acaccctcat gaggacccag 840

gtgatccagg agacctgtga gcgccgcaat cacatcgacc gggctctgga tgccaccgag 900

attctgggca tccttcacag ctgcctcaac cccctcatct acgccttcat tggccagaag 960

tttcgccatg gactcctcaa gattctagct atacatggct tgatcagcaa ggactccctg 1020

cccaaagaca gcaggccttc ctttgttggc tcttcttcag ggcacacttc cactactctc 1080

taa 1083

<210> 53

<211> 355

<212> PRT

<213> Chile person

<400> 53

Met Asp Gln Phe Pro Glu Ser Val Thr Glu Asn Phe Glu Tyr Asp Asp

1 5 10 15

Leu Ala Glu Ala Cys Tyr Ile Gly Asp Ile Val Val Phe Gly Thr Val

20 25 30

Phe Leu Ser Ile Phe Tyr Ser Val Ile Phe Ala Ile Gly Leu Val Gly

35 40 45

Asn Leu Leu Val Val Phe Ala Leu Thr Asn Ser Lys Lys Pro Lys Ser

50 55 60

Val Thr Asp Ile Tyr Leu Leu Asn Leu Ala Leu Ser Asp Leu Leu Phe

65 70 75 80

Val Ala Thr Leu Pro Phe Trp Thr His Tyr Leu Ile Asn Glu Lys Gly

85 90 95

Leu His Asn Ala Met Cys Lys Phe Thr Thr Ala Phe Phe Phe Ile Gly

100 105 110

Phe Phe Gly Ser Ile Phe Phe Ile Thr Val Ile Ser Ile Asp Arg Tyr

115 120 125

Leu Ala Ile Val Leu Ala Ala Asn Ser Met Asn Asn Arg Thr Val Gln

130 135 140

His Gly Val Thr Ile Ser Leu Gly Val Trp Ala Ala Ala Ile Leu Val

145 150 155 160

Ala Ala Pro Gln Phe Met Phe Thr Lys Gln Lys Glu Asn Glu Cys Leu

165 170 175

Gly Asp Tyr Pro Glu Val Leu Gln Glu Ile Trp Pro Val Leu Arg Asn

180 185 190

Val Glu Thr Asn Phe Leu Gly Phe Leu Leu Pro Leu Leu Ile Met Ser

195 200 205

Tyr Cys Tyr Phe Arg Ile Ile Gln Thr Leu Phe Ser Cys Lys Asn His

210 215 220

Lys Lys Ala Lys Ala Ile Lys Leu Ile Leu Leu Val Val Ile Val Phe

225 230 235 240

Phe Leu Phe Trp Thr Pro Tyr Asn Val Met Ile Phe Leu Glu Thr Leu

245 250 255

Lys Leu Tyr Asp Phe Phe Pro Ser Cys Asp Met Arg Lys Asp Leu Arg

260 265 270

Leu Ala Leu Ser Val Thr Glu Thr Val Ala Phe Ser His Cys Cys Leu

275 280 285

Asn Pro Leu Ile Tyr Ala Phe Ala Gly Glu Lys Phe Arg Arg Tyr Leu

290 295 300

Tyr His Leu Tyr Gly Lys Cys Leu Ala Val Leu Cys Gly Arg Ser Val

305 310 315 320

His Val Asp Phe Ser Ser Ser Glu Ser Gln Arg Ser Arg His Gly Ser

325 330 335

Val Leu Ser Ser Asn Phe Thr Tyr His Thr Ser Asp Gly Asp Ala Leu

340 345 350

Leu Leu Leu

355

<210> 54

<211> 1068

<212> DNA

<213> Chile person

<400> 54

atggatcagt tccctgaatc agtgacagaa aactttgagt acgatgattt ggctgaggcc 60

tgttatattg gggacatcgt ggtctttggg actgtgttcc tgtccatatt ctactccgtc 120

atctttgcca ttggcctggt gggaaatttg ttggtagtgt ttgccctcac caacagcaag 180

aagcccaaga gtgtcaccga catttacctc ctgaacctgg ccttgtctga tctgctgttt 240

gtagccactt tgcccttctg gactcactat ttgataaatg aaaagggcct ccacaatgcc 300

atgtgcaaat tcactaccgc cttcttcttc atcggctttt ttggaagcat attcttcatc 360

accgtcatca gcattgatag gtacctggcc atcgtcctgg ccgccaactc catgaacaac 420

cggaccgtgc agcatggcgt caccatcagc ctaggcgtct gggcagcagc cattttggtg 480

gcagcacccc agttcatgtt cacaaagcag aaagaaaatg aatgccttgg tgactacccc 540

gaggtcctcc aggaaatctg gcccgtgctc cgcaatgtgg aaacaaattt tcttggcttc 600

ctactccccc tgctcattat gagttattgc tacttcagaa tcatccagac gctgttttcc 660

tgcaagaacc acaagaaagc caaagccatt aaactgatcc ttctggtggt catcgtgttt 720

ttcctcttct ggacacccta caacgttatg attttcctgg agacgcttaa gctctatgac 780

ttctttccca gttgtgacat gaggaaggat ctgaggctgg ccctcagtgt gactgagacg 840

gttgcattta gccattgttg cctgaatcct ctcatctatg catttgctgg ggagaagttc 900

agaagatacc tttaccacct gtatgggaaa tgcctggctg tcctgtgtgg gcgctcagtc 960

cacgttgatt tctcctcatc tgaatcacaa aggagcaggc atggaagtgt tctgagcagc 1020

aattttactt accacacgag tgatggagat gcattgctcc ttctctga 1068

<210> 55

<211> 373

<212> PRT

<213> Chile person

<400> 55

Met Arg Met Glu Asp Glu Asp Tyr Asn Thr Ser Ile Ser Tyr Gly Asp

1 5 10 15

Glu Tyr Pro Asp Tyr Leu Asp Ser Ile Val Val Leu Glu Asp Leu Ser

20 25 30

Pro Leu Glu Ala Arg Val Thr Arg Ile Phe Leu Val Val Val Tyr Ser

35 40 45

Ile Val Cys Phe Leu Gly Ile Leu Gly Asn Gly Leu Val Ile Ile Ile

50 55 60

Ala Thr Phe Lys Met Lys Lys Thr Val Asn Met Val Trp Phe Leu Asn

65 70 75 80

Leu Ala Val Ala Asp Phe Leu Phe Asn Val Phe Leu Pro Ile His Ile

85 90 95

Thr Tyr Ala Ala Met Asp Tyr His Trp Val Phe Gly Thr Ala Met Cys

100 105 110

Lys Ile Ser Asn Phe Leu Leu Ile His Asn Met Phe Thr Ser Val Phe

115 120 125

Leu Leu Thr Ile Ile Ser Ser Asp Arg Cys Ile Ser Val Leu Leu Pro

130 135 140

Val Trp Ser Gln Asn His Arg Ser Val Arg Leu Ala Tyr Met Ala Cys

145 150 155 160

Met Val Ile Trp Val Leu Ala Phe Phe Leu Ser Ser Pro Ser Leu Val

165 170 175

Phe Arg Asp Thr Ala Asn Leu His Gly Lys Ile Ser Cys Phe Asn Asn

180 185 190

Phe Ser Leu Ser Thr Pro Gly Ser Ser Ser Trp Pro Thr His Ser Gln

195 200 205

Met Asp Pro Val Gly Tyr Ser Arg His Met Val Val Thr Val Thr Arg

210 215 220

Phe Leu Cys Gly Phe Leu Val Pro Val Leu Ile Ile Thr Ala Cys Tyr

225 230 235 240

Leu Thr Ile Val Cys Lys Leu Gln Arg Asn Arg Leu Ala Lys Thr Lys

245 250 255

Lys Pro Phe Lys Ile Ile Val Thr Ile Ile Ile Thr Phe Phe Leu Cys

260 265 270

Trp Cys Pro Tyr His Thr Leu Asn Leu Leu Glu Leu His His Thr Ala

275 280 285

Met Pro Gly Ser Val Phe Ser Leu Gly Leu Pro Leu Ala Thr Ala Leu

290 295 300

Ala Ile Ala Asn Ser Cys Met Asn Pro Ile Leu Tyr Val Phe Met Gly

305 310 315 320

Gln Asp Phe Lys Lys Phe Lys Val Ala Leu Phe Ser Arg Leu Val Asn

325 330 335

Ala Leu Ser Glu Asp Thr Gly His Ser Ser Tyr Pro Ser His Arg Ser

340 345 350

Phe Thr Lys Met Ser Ser Met Asn Glu Arg Thr Ser Met Asn Glu Arg

355 360 365

Glu Thr Gly Met Leu

370

<210> 56

<211> 1122

<212> DNA

<213> Chile person

<400> 56

atgagaatgg aggatgaaga ttacaacact tccatcagtt acggtgatga ataccctgat 60

tatttagact ccattgtggt tttggaggac ttatccccct tggaagccag ggtgaccagg 120

atcttcctgg tggtggtcta cagcatcgtc tgcttcctcg ggattctggg caatggtctg 180

gtgatcatca ttgccacctt caagatgaag aagacagtga acatggtctg gttcctcaac 240

ctggcagtgg cagatttcct gttcaacgtc ttcctcccaa tccatatcac ctatgccgcc 300

atggactacc actgggtttt cgggacagcc atgtgcaaga tcagcaactt ccttctcatc 360

cacaacatgt tcaccagcgt cttcctgctg accatcatca gctctgaccg ctgcatctct 420

gtgctcctcc ctgtctggtc ccagaaccac cgcagcgttc gcctggctta catggcctgc 480

atggtcatct gggtcctggc tttcttcttg agttccccat ctctcgtctt ccgggacaca 540

gccaacctgc atgggaaaat atcctgcttc aacaacttca gcctgtccac acctgggtct 600

tcctcgtggc ccactcactc ccaaatggac cctgtggggt atagccggca catggtggtg 660

actgtcaccc gcttcctctg tggcttcctg gtcccagtcc tcatcatcac agcttgctac 720

ctcaccatcg tgtgcaaact gcagcgcaac cgcctggcca agaccaagaa gcccttcaag 780

attattgtga ccatcatcat taccttcttc ctctgctggt gcccctacca cacactcaac 840

ctcctagagc tccaccacac tgccatgcct ggctctgtct tcagcctggg tttgcccctg 900

gccactgccc ttgccattgc caacagctgc atgaacccca ttctgtatgt tttcatgggt 960

caggacttca agaagttcaa ggtggccctc ttctctcgcc tggtcaatgc tctaagtgaa 1020

gatacaggcc actcttccta ccccagccat agaagcttta ccaagatgtc atcaatgaat 1080

gagaggactt ctatgaatga gagggagacc ggcatgcttt ga 1122

<210> 57

<211> 352

<212> PRT

<213> Chile person

<400> 57

Met Glu Gly Ile Ser Ile Tyr Thr Ser Asp Asn Tyr Thr Glu Glu Met

1 5 10 15

Gly Ser Gly Asp Tyr Asp Ser Met Lys Glu Pro Cys Phe Arg Glu Glu

20 25 30

Asn Ala Asn Phe Asn Lys Ile Phe Leu Pro Thr Ile Tyr Ser Ile Ile

35 40 45

Phe Leu Thr Gly Ile Val Gly Asn Gly Leu Val Ile Leu Val Met Gly

50 55 60

Tyr Gln Lys Lys Leu Arg Ser Met Thr Asp Lys Tyr Arg Leu His Leu

65 70 75 80

Ser Val Ala Asp Leu Leu Phe Val Ile Thr Leu Pro Phe Trp Ala Val

85 90 95

Asp Ala Val Ala Asn Trp Tyr Phe Gly Asn Phe Leu Cys Lys Ala Val

100 105 110

His Val Ile Tyr Thr Val Asn Leu Tyr Ser Ser Val Leu Ile Leu Ala

115 120 125

Phe Ile Ser Leu Asp Arg Tyr Leu Ala Ile Val His Ala Thr Asn Ser

130 135 140

Gln Arg Pro Arg Lys Leu Leu Ala Glu Lys Val Val Tyr Val Gly Val

145 150 155 160

Trp Ile Pro Ala Leu Leu Leu Thr Ile Pro Asp Phe Ile Phe Ala Asn

165 170 175

Val Ser Glu Ala Asp Asp Arg Tyr Ile Cys Asp Arg Phe Tyr Pro Asn

180 185 190

Asp Leu Trp Val Val Val Phe Gln Phe Gln His Ile Met Val Gly Leu

195 200 205

Ile Leu Pro Gly Ile Val Ile Leu Ser Cys Tyr Cys Ile Ile Ile Ser

210 215 220

Lys Leu Ser His Ser Lys Gly His Gln Lys Arg Lys Ala Leu Lys Thr

225 230 235 240

Thr Val Ile Leu Ile Leu Ala Phe Phe Ala Cys Trp Leu Pro Tyr Tyr

245 250 255

Ile Gly Ile Ser Ile Asp Ser Phe Ile Leu Leu Glu Ile Ile Lys Gln

260 265 270

Gly Cys Glu Phe Glu Asn Thr Val His Lys Trp Ile Ser Ile Thr Glu

275 280 285

Ala Leu Ala Phe Phe His Cys Cys Leu Asn Pro Ile Leu Tyr Ala Phe

290 295 300

Leu Gly Ala Lys Phe Lys Thr Ser Ala Gln His Ala Leu Thr Ser Val

305 310 315 320

Ser Arg Gly Ser Ser Leu Lys Ile Leu Ser Lys Gly Lys Arg Gly Gly

325 330 335

His Ser Ser Val Ser Thr Glu Ser Glu Ser Ser Ser Phe His Ser Ser

340 345 350

<210> 58

<211> 1071

<212> DNA

<213> Chile person

<400> 58

atgtccattc ctttgcctct tttgcagata tacacttcag ataactacac cgaggaaatg 60

ggctcagggg actatgactc catgaaggaa ccctgtttcc gtgaagaaaa tgctaatttc 120

aataaaatct tcctgcccac catctactcc atcatcttct taactggcat tgtgggcaat 180

ggattggtca tcctggtcat gggttaccag aagaaactga gaagcatgac ggacaagtac 240

aggctgcacc tgtcagtggc cgacctcctc tttgtcatca cgcttccctt ctgggcagtt 300

gatgccgtgg caaactggta ctttgggaac ttcctatgca aggcagtcca tgtcatctac 360

acagtcaacc tctacagcag tgtcctcatc ctggccttca tcagtctgga ccgctacctg 420

gccatcgtcc acgccaccaa cagtcagagg ccaaggaagc tgttggctga aaaggtggtc 480

tatgttggcg tctggatccc tgccctcctg ctgactattc ccgacttcat ctttgccaac 540

gtcagtgagg cagatgacag atatatctgt gaccgcttct accccaatga cttgtgggtg 600

gttgtgttcc agtttcagca catcatggtt ggccttatcc tgcctggtat tgtcatcctg 660

tcctgctatt gcattatcat ctccaagctg tcacactcca agggccacca gaagcgcaag 720

gccctcaaga ccacagtcat cctcatcctg gctttcttcg cctgttggct gccttactac 780

attgggatca gcatcgactc cttcatcctc ctggaaatca tcaagcaagg gtgtgagttt 840

gagaacactg tgcacaagtg gatttccatc accgaggccc tagctttctt ccactgttgt 900

ctgaacccca tcctctatgc tttccttgga gccaaattta aaacctctgc ccagcacgca 960

ctcacctctg tgagcagagg gtccagcctc aagatcctct ccaaaggaaa gcgaggtgga 1020

cattcatctg tttccactga gtctgagtct tcaagttttc actccagcta a 1071

<210> 59

<211> 352

<212> PRT

<213> Chile person

<400> 59

Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr

1 5 10 15

Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu

20 25 30

Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn

35 40 45

Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met

50 55 60

Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu

65 70 75 80

Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe

85 90 95

Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe

100 105 110

Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu

115 120 125

Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe

130 135 140

Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser

145 150 155 160

Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr

165 170 175

Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn

180 185 190

Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro Leu Leu

195 200 205

Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg Cys

210 215 220

Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile Phe Thr Ile

225 230 235 240

Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr Asn Ile Val Leu Leu

245 250 255

Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu Asn Asn Cys Ser Ser Ser

260 265 270

Asn Arg Leu Asp Gln Ala Met Gln Val Thr Glu Thr Leu Gly Met Thr

275 280 285

His Cys Cys Ile Asn Pro Ile Ile Tyr Ala Phe Val Gly Glu Lys Phe

290 295 300

Arg Asn Tyr Leu Leu Val Phe Phe Gln Lys His Ile Ala Lys Arg Phe

305 310 315 320

Cys Lys Cys Cys Ser Ile Phe Gln Gln Glu Ala Pro Glu Arg Ala Ser

325 330 335

Ser Val Tyr Thr Arg Ser Thr Gly Glu Gln Glu Ile Ser Val Gly Leu

340 345 350

<210> 60

<211> 1059

<212> DNA

<213> Chile person

<400> 60

atggattatc aagtgtcaag tccaatctat gacatcaatt attatacatc ggagccctgc 60

caaaaaatca atgtgaagca aatcgcagcc cgcctcctgc ctccgctcta ctcactggtg 120

ttcatctttg gttttgtggg caacatgctg gtcatcctca tcctgataaa ctgcaaaagg 180

ctgaagagca tgactgacat ctacctgctc aacctggcca tctctgacct gtttttcctt 240

cttactgtcc ccttctgggc tcactatgct gccgcccagt gggactttgg aaatacaatg 300

tgtcaactct tgacagggct ctattttata ggcttcttct ctggaatctt cttcatcatc 360

ctcctgacaa tcgataggta cctggctgtc gtccatgctg tgtttgcttt aaaagccagg 420

acggtcacct ttggggtggt gacaagtgtg atcacttggg tggtggctgt gtttgcgtct 480

ctcccaggaa tcatctttac cagatctcaa aaagaaggtc ttcattacac ctgcagctct 540

cattttccat acagtcagta tcaattctgg aagaatttcc agacattaaa gatagtcatc 600

ttggggctgg tcctgccgct gcttgtcatg gtcatctgct actcgggaat cctaaaaact 660

ctgcttcggt gtcgaaatga gaagaagagg cacagggctg tgaggcttat cttcaccatc 720

atgattgttt attttctctt ctgggctccc tacaacattg tccttctcct gaacaccttc 780

caggaattct ttggcctgaa taattgcagt agctctaaca ggttggacca agctatgcag 840

gtgacagaga ctcttgggat gacgcactgc tgcatcaacc ccatcatcta tgcctttgtc 900

ggggagaagt tcagaaacta cctcttagtc ttcttccaaa agcacattgc caaacgcttc 960

tgcaaatgct gttctatttt ccagcaagag gctcccgagc gagcaagctc agtttacacc 1020

cgatccactg gggagcagga aatatctgtg ggcttgtga 1059

<210> 61

<211> 398

<212> PRT

<213> Chile person

<400> 61

Met Glu Ser Gly Leu Leu Arg Pro Ala Pro Val Ser Glu Val Ile Val

1 5 10 15

Leu His Tyr Asn Tyr Thr Gly Lys Leu Arg Gly Ala Arg Tyr Gln Pro

20 25 30

Gly Ala Gly Leu Arg Ala Asp Ala Val Val Cys Leu Ala Val Cys Ala

35 40 45

Phe Ile Val Leu Glu Asn Leu Ala Val Leu Leu Val Leu Gly Arg His

50 55 60

Pro Arg Phe His Ala Pro Met Phe Leu Leu Leu Gly Ser Leu Thr Leu

65 70 75 80

Ser Asp Leu Leu Ala Gly Ala Ala Tyr Ala Ala Asn Ile Leu Leu Ser

85 90 95

Gly Pro Leu Thr Leu Lys Leu Ser Pro Ala Leu Trp Phe Ala Arg Glu

100 105 110

Gly Gly Val Phe Val Ala Leu Thr Ala Ser Val Leu Ser Leu Leu Ala

115 120 125

Ile Ala Leu Glu Arg Ser Leu Thr Met Ala Arg Arg Gly Pro Ala Pro

130 135 140

Val Ser Ser Arg Gly Arg Thr Leu Ala Met Ala Ala Ala Ala Trp Gly

145 150 155 160

Val Ser Leu Leu Leu Gly Leu Leu Pro Ala Leu Gly Trp Asn Cys Leu

165 170 175

Gly Arg Leu Asp Ala Cys Ser Thr Val Leu Pro Leu Tyr Ala Lys Ala

180 185 190

Tyr Val Leu Phe Cys Val Leu Ala Phe Val Gly Ile Leu Ala Ala Ile

195 200 205

Cys Ala Leu Tyr Ala Arg Ile Tyr Cys Gln Val Arg Ala Asn Ala Arg

210 215 220

Arg Leu Pro Ala Arg Pro Gly Thr Ala Gly Thr Thr Ser Thr Arg Ala

225 230 235 240

Arg Arg Lys Pro Arg Ser Leu Ala Leu Leu Arg Thr Leu Ser Val Val

245 250 255

Leu Leu Ala Phe Val Ala Cys Trp Gly Pro Leu Phe Leu Leu Leu Leu

260 265 270

Leu Asp Val Ala Cys Pro Ala Arg Thr Cys Pro Val Leu Leu Gln Ala

275 280 285

Asp Pro Phe Leu Gly Leu Ala Met Ala Asn Ser Leu Leu Asn Pro Ile

290 295 300

Ile Tyr Thr Leu Thr Asn Arg Asp Leu Arg His Ala Leu Leu Arg Leu

305 310 315 320

Val Cys Cys Gly Arg His Ser Cys Gly Arg Asp Pro Ser Gly Ser Gln

325 330 335

Gln Ser Ala Ser Ala Ala Glu Ala Ser Gly Gly Leu Arg Arg Cys Leu

340 345 350

Pro Pro Gly Leu Asp Gly Ser Phe Ser Gly Ser Glu Arg Ser Ser Pro

355 360 365

Gln Arg Asp Gly Leu Asp Thr Ser Gly Ser Thr Gly Ser Pro Gly Ala

370 375 380

Pro Thr Ala Ala Arg Thr Leu Val Ser Glu Pro Ala Ala Asp

385 390 395

<210> 62

<211> 1197

<212> DNA

<213> Chile person

<400> 62

atggagtcgg ggctgctgcg gccggcgccg gtgagcgagg tcatcgtcct gcattacaac 60

tacaccggca agctccgcgg tgcgcgctac cagccgggtg ccggcctgcg cgccgacgcc 120

gtggtgtgcc tggcggtgtg cgccttcatc gtgctagaga atctagccgt gttgttggtg 180

ctcggacgcc acccgcgctt ccacgctccc atgttcctgc tcctgggcag cctcacgttg 240

tcggatctgc tggcaggcgc cgcctacgcc gccaacatcc tactgtcggg gccgctcacg 300

ctgaaactgt cccccgcgct ctggttcgca cgggagggag gcgtcttcgt ggcactcact 360

gcgtccgtgc tgagcctcct ggccatcgcg ctggagcgca gcctcaccat ggcgcgcagg 420

gggcccgcgc ccgtctccag tcgggggcgc acgctggcga tggcagccgc ggcctggggc 480

gtgtcgctgc tcctcgggct cctgccagcg ctgggctgga attgcctggg tcgcctggac 540

gcttgctcca ctgtcttgcc gctctacgcc aaggcctacg tgctcttctg cgtgctcgcc 600

ttcgtgggca tcctggccgc tatctgtgca ctctacgcgc gcatctactg ccaggtacgc 660

gccaacgcgc ggcgcctgcc ggcacggccc gggactgcgg ggaccacctc gacccgggcg 720

cgtcgcaagc cgcgctcgct ggccttgctg cgcacgctca gcgtggtgct cctggccttt 780

gtggcatgtt ggggccccct cttcctgctg ctgttgctcg acgtggcgtg cccggcgcgc 840

acctgtcctg tactcctgca ggccgatccc ttcctgggac tggccatggc caactcactt 900

ctgaacccca tcatctacac gctcaccaac cgcgacctgc gccacgcgct cctgcgcctg 960

gtctgctgcg gacgccactc ctgcggcaga gacccgagtg gctcccagca gtcggcgagc 1020

gcggctgagg cttccggggg cctgcgccgc tgcctgcccc cgggccttga tgggagcttc 1080

agcggctcgg agcgctcatc gccccagcgc gacgggctgg acaccagcgg ctccacaggc 1140

agccccggtg cacccacagc cgcccggact ctggtatcag aaccggctgc agactga 1197

<210> 63

<211> 951

<212> PRT

<213> Chile person

<400> 63

Gln Pro Ser Val Ser Pro Gly Glu Pro Ser Pro Pro Ser Ile His Pro

1 5 10 15

Gly Lys Ser Asp Leu Ile Val Arg Val Gly Asp Glu Ile Arg Leu Leu

20 25 30

Cys Thr Asp Pro Gly Phe Val Lys Trp Thr Phe Glu Ile Leu Asp Glu

35 40 45

Thr Asn Glu Asn Lys Gln Asn Glu Trp Ile Thr Glu Lys Ala Glu Ala

50 55 60

Thr Asn Thr Gly Lys Tyr Thr Cys Thr Asn Lys His Gly Leu Ser Asn

65 70 75 80

Ser Ile Tyr Val Phe Val Arg Asp Pro Ala Lys Leu Phe Leu Val Asp

85 90 95

Arg Ser Leu Tyr Gly Lys Glu Asp Asn Asp Thr Leu Val Arg Cys Pro

100 105 110

Leu Thr Asp Pro Glu Val Thr Asn Tyr Ser Leu Lys Gly Cys Gln Gly

115 120 125

Lys Pro Leu Pro Lys Asp Leu Arg Phe Ile Pro Asp Pro Lys Ala Gly

130 135 140

Ile Met Ile Lys Ser Val Lys Arg Ala Tyr His Arg Leu Cys Leu His

145 150 155 160

Cys Ser Val Asp Gln Glu Gly Lys Ser Val Leu Ser Glu Lys Phe Ile

165 170 175

Leu Lys Val Arg Pro Ala Phe Lys Ala Val Pro Val Val Ser Val Ser

180 185 190

Lys Ala Ser Tyr Leu Leu Arg Glu Gly Glu Glu Phe Thr Val Thr Cys

195 200 205

Thr Ile Lys Asp Val Ser Ser Ser Val Tyr Ser Thr Trp Lys Arg Glu

210 215 220

Asn Ser Gln Thr Lys Leu Gln Glu Lys Tyr Asn Ser Trp His His Gly

225 230 235 240

Asp Phe Asn Tyr Glu Arg Gln Ala Thr Leu Thr Ile Ser Ser Ala Arg

245 250 255

Val Asn Asp Ser Gly Val Phe Met Cys Tyr Ala Asn Asn Thr Phe Gly

260 265 270

Ser Ala Asn Val Thr Thr Thr Leu Glu Val Val Asp Lys Gly Phe Ile

275 280 285

Asn Ile Phe Pro Met Ile Asn Thr Thr Val Phe Val Asn Asp Gly Glu

290 295 300

Asn Val Asp Leu Ile Val Glu Tyr Glu Ala Phe Pro Lys Pro Glu His

305 310 315 320

Gln Gln Trp Ile Tyr Met Asn Arg Thr Phe Thr Asp Lys Trp Glu Asp

325 330 335

Tyr Pro Lys Ser Glu Asn Glu Ser Asn Ile Arg Tyr Val Ser Glu Leu

340 345 350

His Leu Thr Arg Leu Lys Gly Thr Glu Gly Gly Thr Tyr Thr Phe Leu

355 360 365

Val Ser Asn Ser Asp Val Asn Ala Ala Ile Ala Phe Asn Val Tyr Val

370 375 380

Asn Thr Lys Pro Glu Ile Leu Thr Tyr Asp Arg Leu Val Asn Gly Met

385 390 395 400

Leu Gln Cys Val Ala Ala Gly Phe Pro Glu Pro Thr Ile Asp Trp Tyr

405 410 415

Phe Cys Pro Gly Thr Glu Gln Arg Cys Ser Ala Ser Val Leu Pro Val

420 425 430

Asp Val Gln Thr Leu Asn Ser Ser Gly Pro Pro Phe Gly Lys Leu Val

435 440 445

Val Gln Ser Ser Ile Asp Ser Ser Ala Phe Lys His Asn Gly Thr Val

450 455 460

Glu Cys Lys Ala Tyr Asn Asp Val Gly Lys Thr Ser Ala Tyr Phe Asn

465 470 475 480

Phe Ala Phe Lys Gly Asn Asn Lys Glu Gln Ile His Pro His Thr Leu

485 490 495

Phe Thr Pro Leu Leu Ile Gly Phe Val Ile Val Ala Gly Met Met Cys

500 505 510

Ile Ile Val Met Ile Leu Thr Tyr Lys Tyr Leu Gln Lys Pro Met Tyr

515 520 525

Glu Val Gln Trp Lys Val Val Glu Glu Ile Asn Gly Asn Asn Tyr Val

530 535 540

Tyr Ile Asp Pro Thr Gln Leu Pro Tyr Asp His Lys Trp Glu Phe Pro

545 550 555 560

Arg Asn Arg Leu Ser Phe Gly Lys Thr Leu Gly Ala Gly Ala Phe Gly

565 570 575

Lys Val Val Glu Ala Thr Ala Tyr Gly Leu Ile Lys Ser Asp Ala Ala

580 585 590

Met Thr Val Ala Val Lys Met Leu Lys Pro Ser Ala His Leu Thr Glu

595 600 605

Arg Glu Ala Leu Met Ser Glu Leu Lys Val Leu Ser Tyr Leu Gly Asn

610 615 620

His Met Asn Ile Val Asn Leu Leu Gly Ala Cys Thr Ile Gly Gly Pro

625 630 635 640

Thr Leu Val Ile Thr Glu Tyr Cys Cys Tyr Gly Asp Leu Leu Asn Phe

645 650 655

Leu Arg Arg Lys Arg Asp Ser Phe Ile Cys Ser Lys Gln Glu Asp His

660 665 670

Ala Glu Ala Ala Leu Tyr Lys Asn Leu Leu His Ser Lys Glu Ser Ser

675 680 685

Cys Ser Asp Ser Thr Asn Glu Tyr Met Asp Met Lys Pro Gly Val Ser

690 695 700

Tyr Val Val Pro Thr Lys Ala Asp Lys Arg Arg Ser Val Arg Ile Gly

705 710 715 720

Ser Tyr Ile Glu Arg Asp Val Thr Pro Ala Ile Met Glu Asp Asp Glu

725 730 735

Leu Ala Leu Asp Leu Glu Asp Leu Leu Ser Phe Ser Tyr Gln Val Ala

740 745 750

Lys Gly Met Ala Phe Leu Ala Ser Lys Asn Cys Ile His Arg Asp Leu

755 760 765

Ala Ala Arg Asn Ile Leu Leu Thr His Gly Arg Ile Thr Lys Ile Cys

770 775 780

Asp Phe Gly Leu Ala Arg Asp Ile Lys Asn Asp Ser Asn Tyr Val Val

785 790 795 800

Lys Gly Asn Ala Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile

805 810 815

Phe Asn Cys Val Tyr Thr Phe Glu Ser Asp Val Trp Ser Tyr Gly Ile

820 825 830

Phe Leu Trp Glu Leu Phe Ser Leu Gly Ser Ser Pro Tyr Pro Gly Met

835 840 845

Pro Val Asp Ser Lys Phe Tyr Lys Met Ile Lys Glu Gly Phe Arg Met

850 855 860

Leu Ser Pro Glu His Ala Pro Ala Glu Met Tyr Asp Ile Met Lys Thr

865 870 875 880

Cys Trp Asp Ala Asp Pro Leu Lys Arg Pro Thr Phe Lys Gln Ile Val

885 890 895

Gln Leu Ile Glu Lys Gln Ile Ser Glu Ser Thr Asn His Ile Tyr Ser

900 905 910

Asn Leu Ala Asn Cys Ser Pro Asn Arg Gln Lys Pro Val Val Asp His

915 920 925

Ser Val Arg Ile Asn Ser Val Gly Ser Thr Ala Ser Ser Ser Gln Pro

930 935 940

Leu Leu Val His Asp Asp Val

945 950

<210> 64

<211> 2931

<212> DNA

<213> Chile person

<400> 64

atgagaggcg ctcgcggcgc ctgggatttt ctctgcgttc tgctcctact gcttcgcgtc 60

cagacaggct cttctcaacc atctgtgagt ccaggggaac cgtctccacc atccatccat 120

ccaggaaaat cagacttaat agtccgcgtg ggcgacgaga ttaggctgtt atgcactgat 180

ccgggctttg tcaaatggac ttttgagatc ctggatgaaa cgaatgagaa taagcagaat 240

gaatggatca cggaaaaggc agaagccacc aacaccggca aatacacgtg caccaacaaa 300

cacggcttaa gcaattccat ttatgtgttt gttagagatc ctgccaagct tttccttgtt 360

gaccgctcct tgtatgggaa agaagacaac gacacgctgg tccgctgtcc tctcacagac 420

ccagaagtga ccaattattc cctcaagggg tgccagggga agcctcttcc caaggacttg 480

aggtttattc ctgaccccaa ggcgggcatc atgatcaaaa gtgtgaaacg cgcctaccat 540

cggctctgtc tgcattgttc tgtggaccag gagggcaagt cagtgctgtc ggaaaaattc 600

atcctgaaag tgaggccagc cttcaaagct gtgcctgttg tgtctgtgtc caaagcaagc 660

tatcttctta gggaagggga agaattcaca gtgacgtgca caataaaaga tgtgtctagt 720

tctgtgtact caacgtggaa aagagaaaac agtcagacta aactacagga gaaatataat 780

agctggcatc acggtgactt caattatgaa cgtcaggcaa cgttgactat cagttcagcg 840

agagttaatg attctggagt gttcatgtgt tatgccaata atacttttgg atcagcaaat 900

gtcacaacaa ccttggaagt agtagataaa ggattcatta atatcttccc catgataaac 960

actacagtat ttgtaaacga tggagaaaat gtagatttga ttgttgaata tgaagcattc 1020

cccaaacctg aacaccagca gtggatctat atgaacagaa ccttcactga taaatgggaa 1080

gattatccca agtctgagaa tgaaagtaat atcagatacg taagtgaact tcatctaacg 1140

agattaaaag gcaccgaagg aggcacttac acattcctag tgtccaattc tgacgtcaat 1200

gctgccatag catttaatgt ttatgtgaat acaaaaccag aaatcctgac ttacgacagg 1260

ctcgtgaatg gcatgctcca atgtgtggca gcaggattcc cagagcccac aatagattgg 1320

tatttttgtc caggaactga gcagagatgc tctgcttctg tactgccagt ggatgtgcag 1380

acactaaact catctgggcc accgtttgga aagctagtgg ttcagagttc tatagattct 1440

agtgcattca agcacaatgg cacggttgaa tgtaaggctt acaacgatgt gggcaagact 1500

tctgcctatt ttaactttgc atttaaaggt aacaacaaag agcaaatcca tccccacacc 1560

ctgttcactc ctttgctgat tggtttcgta atcgtagctg gcatgatgtg cattattgtg 1620

atgattctga cctacaaata tttacagaaa cccatgtatg aagtacagtg gaaggttgtt 1680

gaggagataa atggaaacaa ttatgtttac atagacccaa cacaacttcc ttatgatcac 1740

aaatgggagt ttcccagaaa caggctgagt tttgggaaaa ccctgggtgc tggagctttc 1800

gggaaggttg ttgaggcaac tgcttatggc ttaattaagt cagatgcggc catgactgtc 1860

gctgtaaaga tgctcaagcc gagtgcccat ttgacagaac gggaagccct catgtctgaa 1920

ctcaaagtcc tgagttacct tggtaatcac atgaatattg tgaatctact tggagcctgc 1980

accattggag ggcccaccct ggtcattaca gaatattgtt gctatggtga tcttttgaat 2040

tttttgagaa gaaaacgtga ttcatttatt tgttcaaagc aggaagatca tgcagaagct 2100

gcactttata agaatcttct gcattcaaag gagtcttcct gcagcgatag tactaatgag 2160

tacatggaca tgaaacctgg agtttcttat gttgtcccaa ccaaggccga caaaaggaga 2220

tctgtgagaa taggctcata catagaaaga gatgtgactc ccgccatcat ggaggatgac 2280

gagttggccc tagacttaga agacttgctg agcttttctt accaggtggc aaagggcatg 2340

gctttcctcg cctccaagaa ttgtattcac agagacttgg cagccagaaa tatcctcctt 2400

actcatggtc ggatcacaaa gatttgtgat tttggtctag ccagagacat caagaatgat 2460

tctaattatg tggttaaagg aaacgctcga ctacctgtga agtggatggc acctgaaagc 2520

attttcaact gtgtatacac gtttgaaagt gacgtctggt cctatgggat ttttctttgg 2580

gagctgttct ctttaggaag cagcccctat cctggaatgc cggtcgattc taagttctac 2640

aagatgatca aggaaggctt ccggatgctc agccctgaac acgcacctgc tgaaatgtat 2700

gacataatga agacttgctg ggatgcagat cccctaaaaa gaccaacatt caagcaaatt 2760

gttcagctaa ttgagaagca gatttcagag agcaccaatc atatttactc caacttagca 2820

aactgcagcc ccaaccgaca gaagcccgtg gtagaccatt ctgtgcggat caattctgtc 2880

ggcagcaccg cttcctcctc ccagcctctg cttgtgcacg acgatgtctg a 2931

<210> 65

<211> 2549

<212> PRT

<213> Chile person

<400> 65

Met Leu Gly Thr Gly Pro Ala Ala Ala Thr Thr Ala Ala Thr Thr Ser

1 5 10 15

Ser Asn Val Ser Val Leu Gln Gln Phe Ala Ser Gly Leu Lys Ser Arg

20 25 30

Asn Glu Glu Thr Arg Ala Lys Ala Ala Lys Glu Leu Gln His Tyr Val

35 40 45

Thr Met Glu Leu Arg Glu Met Ser Gln Glu Glu Ser Thr Arg Phe Tyr

50 55 60

Asp Gln Leu Asn His His Ile Phe Glu Leu Val Ser Ser Ser Asp Ala

65 70 75 80

Asn Glu Arg Lys Gly Gly Ile Leu Ala Ile Ala Ser Leu Ile Gly Val

85 90 95

Glu Gly Gly Asn Ala Thr Arg Ile Gly Arg Phe Ala Asn Tyr Leu Arg

100 105 110

Asn Leu Leu Pro Ser Asn Asp Pro Val Val Met Glu Met Ala Ser Lys

115 120 125

Ala Ile Gly Arg Leu Ala Met Ala Gly Asp Thr Phe Thr Ala Glu Tyr

130 135 140

Val Glu Phe Glu Val Lys Arg Ala Leu Glu Trp Leu Gly Ala Asp Arg

145 150 155 160

Asn Glu Gly Arg Arg His Ala Ala Val Leu Val Leu Arg Glu Leu Ala

165 170 175

Ile Ser Val Pro Thr Phe Phe Phe Gln Gln Val Gln Pro Phe Phe Asp

180 185 190

Asn Ile Phe Val Ala Val Trp Asp Pro Lys Gln Ala Ile Arg Glu Gly

195 200 205

Ala Val Ala Ala Leu Arg Ala Cys Leu Ile Leu Thr Thr Gln Arg Glu

210 215 220

Pro Lys Glu Met Gln Lys Pro Gln Trp Tyr Arg His Thr Phe Glu Glu

225 230 235 240

Ala Glu Lys Gly Phe Asp Glu Thr Leu Ala Lys Glu Lys Gly Met Asn

245 250 255

Arg Asp Asp Arg Ile His Gly Ala Leu Leu Ile Leu Asn Glu Leu Val

260 265 270

Arg Ile Ser Ser Met Glu Gly Glu Arg Leu Arg Glu Glu Met Glu Glu

275 280 285

Ile Thr Gln Gln Gln Leu Val His Asp Lys Tyr Cys Lys Asp Leu Met

290 295 300

Gly Phe Gly Thr Lys Pro Arg His Ile Thr Pro Phe Thr Ser Phe Gln

305 310 315 320

Ala Val Gln Pro Gln Gln Ser Asn Ala Leu Val Gly Leu Leu Gly Tyr

325 330 335

Ser Ser His Gln Gly Leu Met Gly Phe Gly Thr Ser Pro Ser Pro Ala

340 345 350

Lys Ser Thr Leu Val Glu Ser Arg Cys Cys Arg Asp Leu Met Glu Glu

355 360 365

Lys Phe Asp Gln Val Cys Gln Trp Val Leu Lys Cys Arg Asn Ser Lys

370 375 380

Asn Ser Leu Ile Gln Met Thr Ile Leu Asn Leu Leu Pro Arg Leu Ala

385 390 395 400

Ala Phe Arg Pro Ser Ala Phe Thr Asp Thr Gln Tyr Leu Gln Asp Thr

405 410 415

Met Asn His Val Leu Ser Cys Val Lys Lys Glu Lys Glu Arg Thr Ala

420 425 430

Ala Phe Gln Ala Leu Gly Leu Leu Ser Val Ala Val Arg Ser Glu Phe

435 440 445

Lys Val Tyr Leu Pro Arg Val Leu Asp Ile Ile Arg Ala Ala Leu Pro

450 455 460

Pro Lys Asp Phe Ala His Lys Arg Gln Lys Ala Met Gln Val Asp Ala

465 470 475 480

Thr Val Phe Thr Cys Ile Ser Met Leu Ala Arg Ala Met Gly Pro Gly

485 490 495

Ile Gln Gln Asp Ile Lys Glu Leu Leu Glu Pro Met Leu Ala Val Gly

500 505 510

Leu Ser Pro Ala Leu Thr Ala Val Leu Tyr Asp Leu Ser Arg Gln Ile

515 520 525

Pro Gln Leu Lys Lys Asp Ile Gln Asp Gly Leu Leu Lys Met Leu Ser

530 535 540

Leu Val Leu Met His Lys Pro Leu Arg His Pro Gly Met Pro Lys Gly

545 550 555 560

Leu Ala His Gln Leu Ala Ser Pro Gly Leu Thr Thr Leu Pro Glu Ala

565 570 575

Ser Asp Val Gly Ser Ile Thr Leu Ala Leu Arg Thr Leu Gly Ser Phe

580 585 590

Glu Phe Glu Gly His Ser Leu Thr Gln Phe Val Arg His Cys Ala Asp

595 600 605

His Phe Leu Asn Ser Glu His Lys Glu Ile Arg Met Glu Ala Ala Arg

610 615 620

Thr Cys Ser Arg Leu Leu Thr Pro Ser Ile His Leu Ile Ser Gly His

625 630 635 640

Ala His Val Val Ser Gln Thr Ala Val Gln Val Val Ala Asp Val Leu

645 650 655

Ser Lys Leu Leu Val Val Gly Ile Thr Asp Pro Asp Pro Asp Ile Arg

660 665 670

Tyr Cys Val Leu Ala Ser Leu Asp Glu Arg Phe Asp Ala His Leu Ala

675 680 685

Gln Ala Glu Asn Leu Gln Ala Leu Phe Val Ala Leu Asn Asp Gln Val

690 695 700

Phe Glu Ile Arg Glu Leu Ala Ile Cys Thr Val Gly Arg Leu Ser Ser

705 710 715 720

Met Asn Pro Ala Phe Val Met Pro Phe Leu Arg Lys Met Leu Ile Gln

725 730 735

Ile Leu Thr Glu Leu Glu His Ser Gly Ile Gly Arg Ile Lys Glu Gln

740 745 750

Ser Ala Arg Met Leu Gly His Leu Val Ser Asn Ala Pro Arg Leu Ile

755 760 765

Arg Pro Tyr Met Glu Pro Ile Leu Lys Ala Leu Ile Leu Lys Leu Lys

770 775 780

Asp Pro Asp Pro Asp Pro Asn Pro Gly Val Ile Asn Asn Val Leu Ala

785 790 795 800

Thr Ile Gly Glu Leu Ala Gln Val Ser Gly Leu Glu Met Arg Lys Trp

805 810 815

Val Asp Glu Leu Phe Ile Ile Ile Met Asp Met Leu Gln Asp Ser Ser

820 825 830

Leu Leu Ala Lys Arg Gln Val Ala Leu Trp Thr Leu Gly Gln Leu Val

835 840 845

Ala Ser Thr Gly Tyr Val Val Glu Pro Tyr Arg Lys Tyr Pro Thr Leu

850 855 860

Leu Glu Val Leu Leu Asn Phe Leu Lys Thr Glu Gln Asn Gln Gly Thr

865 870 875 880

Arg Arg Glu Ala Ile Arg Val Leu Gly Leu Leu Gly Ala Leu Asp Pro

885 890 895

Tyr Lys His Lys Val Asn Ile Gly Met Ile Asp Gln Ser Arg Asp Ala

900 905 910

Ser Ala Val Ser Leu Ser Glu Ser Lys Ser Ser Gln Asp Ser Ser Asp

915 920 925

Tyr Ser Thr Ser Glu Met Leu Val Asn Met Gly Asn Leu Pro Leu Asp

930 935 940

Glu Phe Tyr Pro Ala Val Ser Met Val Ala Leu Met Arg Ile Phe Arg

945 950 955 960

Asp Gln Ser Leu Ser His His His Thr Met Val Val Gln Ala Ile Thr

965 970 975

Phe Ile Phe Lys Ser Leu Gly Leu Lys Cys Val Gln Phe Leu Pro Gln

980 985 990

Val Met Pro Thr Phe Leu Asn Val Ile Arg Val Cys Asp Gly Ala Ile

995 1000 1005

Arg Glu Phe Leu Phe Gln Gln Leu Gly Met Leu Val Ser Phe Val

1010 1015 1020

Lys Ser His Ile Arg Pro Tyr Met Asp Glu Ile Val Thr Leu Met

1025 1030 1035

Arg Glu Phe Trp Val Met Asn Thr Ser Ile Gln Ser Thr Ile Ile

1040 1045 1050

Leu Leu Ile Glu Gln Ile Val Val Ala Leu Gly Gly Glu Phe Lys

1055 1060 1065

Leu Tyr Leu Pro Gln Leu Ile Pro His Met Leu Arg Val Phe Met

1070 1075 1080

His Asp Asn Ser Pro Gly Arg Ile Val Ser Ile Lys Leu Leu Ala

1085 1090 1095

Ala Ile Gln Leu Phe Gly Ala Asn Leu Asp Asp Tyr Leu His Leu

1100 1105 1110

Leu Leu Pro Pro Ile Val Lys Leu Phe Asp Ala Pro Glu Ala Pro

1115 1120 1125

Leu Pro Ser Arg Lys Ala Ala Leu Glu Thr Val Asp Arg Leu Thr

1130 1135 1140

Glu Ser Leu Asp Phe Thr Asp Tyr Ala Ser Arg Ile Ile His Pro

1145 1150 1155

Ile Val Arg Thr Leu Asp Gln Ser Pro Glu Leu Arg Ser Thr Ala

1160 1165 1170

Met Asp Thr Leu Ser Ser Leu Val Phe Gln Leu Gly Lys Lys Tyr

1175 1180 1185

Gln Ile Phe Ile Pro Met Val Asn Lys Val Leu Val Arg His Arg

1190 1195 1200

Ile Asn His Gln Arg Tyr Asp Val Leu Ile Cys Arg Ile Val Lys

1205 1210 1215

Gly Tyr Thr Leu Ala Asp Glu Glu Glu Asp Pro Leu Ile Tyr Gln

1220 1225 1230

His Arg Met Leu Arg Ser Gly Gln Gly Asp Ala Leu Ala Ser Gly

1235 1240 1245

Pro Val Glu Thr Gly Pro Met Lys Lys Leu His Val Ser Thr Ile

1250 1255 1260

Asn Leu Gln Lys Ala Trp Gly Ala Ala Arg Arg Val Ser Lys Asp

1265 1270 1275

Asp Trp Leu Glu Trp Leu Arg Arg Leu Ser Leu Glu Leu Leu Lys

1280 1285 1290

Asp Ser Ser Ser Pro Ser Leu Arg Ser Cys Trp Ala Leu Ala Gln

1295 1300 1305

Ala Tyr Asn Pro Met Ala Arg Asp Leu Phe Asn Ala Ala Phe Val

1310 1315 1320

Ser Cys Trp Ser Glu Leu Asn Glu Asp Gln Gln Asp Glu Leu Ile

1325 1330 1335

Arg Ser Ile Glu Leu Ala Leu Thr Ser Gln Asp Ile Ala Glu Val

1340 1345 1350

Thr Gln Thr Leu Leu Asn Leu Ala Glu Phe Met Glu His Ser Asp

1355 1360 1365

Lys Gly Pro Leu Pro Leu Arg Asp Asp Asn Gly Ile Val Leu Leu

1370 1375 1380

Gly Glu Arg Ala Ala Lys Cys Arg Ala Tyr Ala Lys Ala Leu His

1385 1390 1395

Tyr Lys Glu Leu Glu Phe Gln Lys Gly Pro Thr Pro Ala Ile Leu

1400 1405 1410

Glu Ser Leu Ile Ser Ile Asn Asn Lys Leu Gln Gln Pro Glu Ala

1415 1420 1425

Ala Ala Gly Val Leu Glu Tyr Ala Met Lys His Phe Gly Glu Leu

1430 1435 1440

Glu Ile Gln Ala Thr Trp Tyr Glu Lys Leu His Glu Trp Glu Asp

1445 1450 1455

Ala Leu Val Ala Tyr Asp Lys Lys Met Asp Thr Asn Lys Asp Asp

1460 1465 1470

Pro Glu Leu Met Leu Gly Arg Met Arg Cys Leu Glu Ala Leu Gly

1475 1480 1485

Glu Trp Gly Gln Leu His Gln Gln Cys Cys Glu Lys Trp Thr Leu

1490 1495 1500

Val Asn Asp Glu Thr Gln Ala Lys Met Ala Arg Met Ala Ala Ala

1505 1510 1515

Ala Ala Trp Gly Leu Gly Gln Trp Asp Ser Met Glu Glu Tyr Thr

1520 1525 1530

Cys Met Ile Pro Arg Asp Thr His Asp Gly Ala Phe Tyr Arg Ala

1535 1540 1545

Val Leu Ala Leu His Gln Asp Leu Phe Ser Leu Ala Gln Gln Cys

1550 1555 1560

Ile Asp Lys Ala Arg Asp Leu Leu Asp Ala Glu Leu Thr Ala Met

1565 1570 1575

Ala Gly Glu Ser Tyr Ser Arg Ala Tyr Gly Ala Met Val Ser Cys

1580 1585 1590

His Met Leu Ser Glu Leu Glu Glu Val Ile Gln Tyr Lys Leu Val

1595 1600 1605

Pro Glu Arg Arg Glu Ile Ile Arg Gln Ile Trp Trp Glu Arg Leu

1610 1615 1620

Gln Gly Cys Gln Arg Ile Val Glu Asp Trp Gln Lys Ile Leu Met

1625 1630 1635

Val Arg Ser Leu Val Val Ser Pro His Glu Asp Met Arg Thr Trp

1640 1645 1650

Leu Lys Tyr Ala Ser Leu Cys Gly Lys Ser Gly Arg Leu Ala Leu

1655 1660 1665

Ala His Lys Thr Leu Val Leu Leu Leu Gly Val Asp Pro Ser Arg

1670 1675 1680

Gln Leu Asp His Pro Leu Pro Thr Val His Pro Gln Val Thr Tyr

1685 1690 1695

Ala Tyr Met Lys Asn Met Trp Lys Ser Ala Arg Lys Ile Asp Ala

1700 1705 1710

Phe Gln His Met Gln His Phe Val Gln Thr Met Gln Gln Gln Ala

1715 1720 1725

Gln His Ala Ile Ala Thr Glu Asp Gln Gln His Lys Gln Glu Leu

1730 1735 1740

His Lys Leu Met Ala Arg Cys Phe Leu Lys Leu Gly Glu Trp Gln

1745 1750 1755

Leu Asn Leu Gln Gly Ile Asn Glu Ser Thr Ile Pro Lys Val Leu

1760 1765 1770

Gln Tyr Tyr Ser Ala Ala Thr Glu His Asp Arg Ser Trp Tyr Lys

1775 1780 1785

Ala Trp His Ala Trp Ala Val Met Asn Phe Glu Ala Val Leu His

1790 1795 1800

Tyr Lys His Gln Asn Gln Ala Arg Asp Glu Lys Lys Lys Leu Arg

1805 1810 1815

His Ala Ser Gly Ala Asn Ile Thr Asn Ala Thr Thr Ala Ala Thr

1820 1825 1830

Thr Ala Ala Thr Ala Thr Thr Thr Ala Ser Thr Glu Gly Ser Asn

1835 1840 1845

Ser Glu Ser Glu Ala Glu Ser Thr Glu Asn Ser Pro Thr Pro Ser

1850 1855 1860

Pro Leu Gln Lys Lys Val Thr Glu Asp Leu Ser Lys Thr Leu Leu

1865 1870 1875

Met Tyr Thr Val Pro Ala Val Gln Gly Phe Phe Arg Ser Ile Ser

1880 1885 1890

Leu Ser Arg Gly Asn Asn Leu Gln Asp Thr Leu Arg Val Leu Thr

1895 1900 1905

Leu Trp Phe Asp Tyr Gly His Trp Pro Asp Val Asn Glu Ala Leu

1910 1915 1920

Val Glu Gly Val Lys Ala Ile Gln Ile Asp Thr Trp Leu Gln Val

1925 1930 1935

Ile Pro Gln Leu Ile Ala Arg Ile Asp Thr Pro Arg Pro Leu Val

1940 1945 1950

Gly Arg Leu Ile His Gln Leu Leu Thr Asp Ile Gly Arg Tyr His

1955 1960 1965

Pro Gln Ala Leu Ile Tyr Pro Leu Thr Val Ala Ser Lys Ser Thr

1970 1975 1980

Thr Thr Ala Arg His Asn Ala Ala Asn Lys Ile Leu Lys Asn Met

1985 1990 1995

Cys Glu His Ser Asn Thr Leu Val Gln Gln Ala Met Met Val Ser

2000 2005 2010

Glu Glu Leu Ile Arg Val Ala Ile Leu Trp His Glu Met Trp His

2015 2020 2025

Glu Gly Leu Glu Glu Ala Ser Arg Leu Tyr Phe Gly Glu Arg Asn

2030 2035 2040

Val Lys Gly Met Phe Glu Val Leu Glu Pro Leu His Ala Met Met

2045 2050 2055

Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr Ser Phe Asn Gln Ala

2060 2065 2070

Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp Cys Arg Lys Tyr

2075 2080 2085

Met Lys Ser Gly Asn Val Lys Asp Leu Thr Gln Ala Trp Asp Leu

2090 2095 2100

Tyr Tyr His Val Phe Arg Arg Ile Ser Lys Gln Leu Pro Gln Leu

2105 2110 2115

Thr Ser Leu Glu Leu Gln Tyr Val Ser Pro Lys Leu Leu Met Cys

2120 2125 2130

Arg Asp Leu Glu Leu Ala Val Pro Gly Thr Tyr Asp Pro Asn Gln

2135 2140 2145

Pro Ile Ile Arg Ile Gln Ser Ile Ala Pro Ser Leu Gln Val Ile

2150 2155 2160

Thr Ser Lys Gln Arg Pro Arg Lys Leu Thr Leu Met Gly Ser Asn

2165 2170 2175

Gly His Glu Phe Val Phe Leu Leu Lys Gly His Glu Asp Leu Arg

2180 2185 2190

Gln Asp Glu Arg Val Met Gln Leu Phe Gly Leu Val Asn Thr Leu

2195 2200 2205

Leu Ala Asn Asp Pro Thr Ser Leu Arg Lys Asn Leu Ser Ile Gln

2210 2215 2220

Arg Tyr Ala Val Ile Pro Leu Ser Thr Asn Ser Gly Leu Ile Gly

2225 2230 2235

Trp Val Pro His Cys Asp Thr Leu His Ala Leu Ile Arg Asp Tyr

2240 2245 2250

Arg Glu Lys Lys Lys Ile Leu Leu Asn Ile Glu His Arg Ile Met

2255 2260 2265

Leu Arg Met Ala Pro Asp Tyr Asp His Leu Thr Leu Met Gln Lys

2270 2275 2280

Val Glu Val Phe Glu His Ala Val Asn Asn Thr Ala Gly Asp Asp

2285 2290 2295

Leu Ala Lys Leu Leu Trp Leu Lys Ser Pro Ser Ser Glu Val Trp

2300 2305 2310

Phe Asp Arg Arg Thr Asn Tyr Thr Arg Ser Leu Ala Val Met Ser

2315 2320 2325

Met Val Gly Tyr Ile Leu Gly Leu Gly Asp Arg His Pro Ser Asn

2330 2335 2340

Leu Met Leu Asp Arg Leu Ser Gly Lys Ile Leu His Ile Asp Phe

2345 2350 2355

Gly Asp Cys Phe Glu Val Ala Met Thr Arg Glu Lys Phe Pro Glu

2360 2365 2370

Lys Ile Pro Phe Arg Leu Thr Arg Met Leu Thr Asn Ala Met Glu

2375 2380 2385

Val Thr Gly Leu Asp Gly Asn Tyr Arg Ile Thr Cys His Thr Val

2390 2395 2400

Met Glu Val Leu Arg Glu His Lys Asp Ser Val Met Ala Val Leu

2405 2410 2415

Glu Ala Phe Val Tyr Asp Pro Leu Leu Asn Trp Arg Leu Met Asp

2420 2425 2430

Thr Asn Thr Lys Gly Asn Lys Arg Ser Arg Thr Arg Thr Asp Ser

2435 2440 2445

Tyr Ser Ala Gly Gln Ser Val Glu Ile Leu Asp Gly Val Glu Leu

2450 2455 2460

Gly Glu Pro Ala His Lys Lys Thr Gly Thr Thr Val Pro Glu Ser

2465 2470 2475

Ile His Ser Phe Ile Gly Asp Gly Leu Val Lys Pro Glu Ala Leu

2480 2485 2490

Asn Lys Lys Ala Ile Gln Ile Ile Asn Arg Val Arg Asp Lys Leu

2495 2500 2505

Thr Gly Arg Asp Phe Ser His Asp Asp Thr Leu Asp Val Pro Thr

2510 2515 2520

Gln Val Glu Leu Leu Ile Lys Gln Ala Thr Ser His Glu Asn Leu

2525 2530 2535

Cys Gln Cys Tyr Ile Gly Trp Cys Pro Phe Trp

2540 2545

<210> 66

<211> 7650

<212> DNA

<213> Chile person

<400> 66

atgcttggaa ccggacctgc cgccgccacc accgctgcca ccacatctag caatgtgagc 60

gtcctgcagc agtttgccag tggcctaaag agccggaatg aggaaaccag ggccaaagcc 120

gccaaggagc tccagcacta tgtcaccatg gaactccgag agatgagtca agaggagtct 180

actcgcttct atgaccaact gaaccatcac atttttgaat tggtttccag ctcagatgcc 240

aatgagagga aaggtggcat cttggccata gctagcctca taggagtgga aggtgggaat 300

gccacccgaa ttggcagatt tgccaactat cttcggaacc tcctcccctc caatgaccca 360

gttgtcatgg aaatggcatc caaggccatt ggccgtcttg ccatggcagg ggacactttt 420

accgctgagt acgtggaatt tgaggtgaag cgagccctgg aatggctggg tgctgaccgc 480

aatgagggcc ggagacatgc agctgtcctg gttctccgtg agctggccat cagcgtccct 540

accttcttct tccagcaagt gcaacccttc tttgacaaca tttttgtggc cgtgtgggac 600

cccaaacagg ccatccgtga gggagctgta gccgcccttc gtgcctgtct gattctcaca 660

acccagcgtg agccgaagga gatgcagaag cctcagtggt acaggcacac atttgaagaa 720

gcagagaagg gatttgatga gaccttggcc aaagagaagg gcatgaatcg ggatgatcgg 780

atccatggag ccttgttgat ccttaacgag ctggtccgaa tcagcagcat ggagggagag 840

cgtctgagag aagaaatgga agaaatcaca cagcagcagc tggtacacga caagtactgc 900

aaagatctca tgggcttcgg aacaaaacct cgtcacatta cccccttcac cagtttccag 960

gctgtacagc cccagcagtc aaatgccttg gtggggctgc tggggtacag ctctcaccaa 1020

ggcctcatgg gatttgggac ctcccccagt ccagctaagt ccaccctggt ggagagccgg 1080

tgttgcagag acttgatgga ggagaaattt gatcaggtgt gccagtgggt gctgaaatgc 1140

aggaatagca agaactcgct gatccaaatg acaatcctta atttgttgcc ccgcttggct 1200

gcattccgac cttctgcctt cacagatacc cagtatctcc aagataccat gaaccatgtc 1260

ctaagctgtg tcaagaagga gaaggaacgt acagcggcct tccaagccct ggggctactt 1320

tctgtggctg tgaggtctga gtttaaggtc tatttgcctc gcgtgctgga catcatccga 1380

gcggccctgc ccccaaagga cttcgcccat aagaggcaga aggcaatgca ggtggatgcc 1440

acagtcttca cttgcatcag catgctggct cgagcaatgg ggccaggcat ccagcaggat 1500

atcaaggagc tgctggagcc catgctggca gtgggactaa gccctgccct cactgcagtg 1560

ctctacgacc tgagccgtca gattccacag ctaaagaagg acattcaaga tgggctactg 1620

aaaatgctgt ccctggtcct tatgcacaaa ccccttcgcc acccaggcat gcccaagggc 1680

ctggcccatc agctggcctc tcctggcctc acgaccctcc ctgaggccag cgatgtgggc 1740

agcatcactc ttgccctccg aacgcttggc agctttgaat ttgaaggcca ctctctgacc 1800

caatttgttc gccactgtgc ggatcatttc ctgaacagtg agcacaagga gatccgcatg 1860

gaggctgccc gcacctgctc ccgcctgctc acaccctcca tccacctcat cagtggccat 1920

gctcatgtgg ttagccagac cgcagtgcaa gtggtggcag atgtgcttag caaactgctc 1980

gtagttggga taacagatcc tgaccctgac attcgctact gtgtcttggc gtccctggac 2040

gagcgctttg atgcacacct ggcccaggcg gagaacttgc aggccttgtt tgtggctctg 2100

aatgaccagg tgtttgagat ccgggagctg gccatctgca ctgtgggccg actcagtagc 2160

atgaaccctg cctttgtcat gcctttcctg cgcaagatgc tcatccagat tttgacagag 2220

ttggagcaca gtgggattgg aagaatcaaa gagcagagtg cccgcatgct ggggcacctg 2280

gtctccaatg ccccccgact catccgcccc tacatggagc ctattctgaa ggcattaatt 2340

ttgaaactga aagatccaga ccctgatcca aacccaggtg tgatcaataa tgtcctggca 2400

acaataggag aattggcaca ggttagtggc ctggaaatga ggaaatgggt tgatgaactt 2460

tttattatca tcatggacat gctccaggat tcctctttgt tggccaaaag gcaggtggct 2520

ctgtggaccc tgggacagtt ggtggccagc actggctatg tagtagagcc ctacaggaag 2580

taccctactt tgcttgaggt gctactgaat tttctgaaga ctgagcagaa ccagggtaca 2640

cgcagagagg ccatccgtgt gttagggctt ttaggggctt tggatcctta caagcacaaa 2700

gtgaacattg gcatgataga ccagtcccgg gatgcctctg ctgtcagcct gtcagaatcc 2760

aagtcaagtc aggattcctc tgactatagc actagtgaaa tgctggtcaa catgggaaac 2820

ttgcctctgg atgagttcta cccagctgtg tccatggtgg ccctgatgcg gatcttccga 2880

gaccagtcac tctctcatca tcacaccatg gttgtccagg ccatcacctt catcttcaag 2940

tccctgggac tcaaatgtgt gcagttcctg ccccaggtca tgcccacgtt ccttaacgtc 3000

attcgagtct gtgatggggc catccgggaa tttttgttcc agcagctggg aatgttggtg 3060

tcctttgtga agagccacat cagaccttat atggatgaaa tagtcaccct catgagagaa 3120

ttctgggtca tgaacacctc aattcagagc acgatcattc ttctcattga gcaaattgtg 3180

gtagctcttg ggggtgaatt taagctctac ctgccccagc tgatcccaca catgctgcgt 3240

gtcttcatgc atgacaacag cccaggccgc attgtctcta tcaagttact ggctgcaatc 3300

cagctgtttg gcgccaacct ggatgactac ctgcatttac tgctgcctcc tattgttaag 3360

ttgtttgatg cccctgaagc tccactgcca tctcgaaagg cagcgctaga gactgtggac 3420

cgcctgacgg agtccctgga tttcactgac tatgcctccc ggatcattca ccctattgtt 3480

cgaacactgg accagagccc agaactgcgc tccacagcca tggacacgct gtcttcactt 3540

gtttttcagc tggggaagaa gtaccaaatt ttcattccaa tggtgaataa agttctggtg 3600

cgacaccgaa tcaatcatca gcgctatgat gtgctcatct gcagaattgt caagggatac 3660

acacttgctg atgaagagga ggatcctttg atttaccagc atcggatgct taggagtggc 3720

caaggggatg cattggctag tggaccagtg gaaacaggac ccatgaagaa actgcacgtc 3780

agcaccatca acctccaaaa ggcctggggc gctgccagga gggtctccaa agatgactgg 3840

ctggaatggc tgagacggct gagcctggag ctgctgaagg actcatcatc gccctccctg 3900

cgctcctgct gggccctggc acaggcctac aacccgatgg ccagggatct cttcaatgct 3960

gcatttgtgt cctgctggtc tgaactgaat gaagatcaac aggatgagct catcagaagc 4020

atcgagttgg ccctcacctc acaagacatc gctgaagtca cacagaccct cttaaacttg 4080

gctgaattca tggaacacag tgacaagggc cccctgccac tgagagatga caatggcatt 4140

gttctgctgg gtgagagagc tgccaagtgc cgagcatatg ccaaagcact acactacaaa 4200

gaactggagt tccagaaagg ccccacccct gccattctag aatctctcat cagcattaat 4260

aataagctac agcagccgga ggcagcggcc ggagtgttag aatatgccat gaaacacttt 4320

ggagagctgg agatccaggc tacctggtat gagaaactgc acgagtggga ggatgccctt 4380

gtggcctatg acaagaaaat ggacaccaac aaggacgacc cagagctgat gctgggccgc 4440

atgcgctgcc tcgaggcctt gggggaatgg ggtcaactcc accagcagtg ctgtgaaaag 4500

tggaccctgg ttaatgatga gacccaagcc aagatggccc ggatggctgc tgcagctgca 4560

tggggtttag gtcagtggga cagcatggaa gaatacacct gtatgatccc tcgggacacc 4620

catgatgggg cattttatag agctgtgctg gcactgcatc aggacctctt ctccttggca 4680

caacagtgca ttgacaaggc cagggacctg ctggatgctg aattaactgc gatggcagga 4740

gagagttaca gtcgggcata tggggccatg gtttcttgcc acatgctgtc cgagctggag 4800

gaggttatcc agtacaaact tgtccccgag cgacgagaga tcatccgcca gatctggtgg 4860

gagagactgc agggctgcca gcgtatcgta gaggactggc agaaaatcct tatggtgcgg 4920

tcccttgtgg tcagccctca tgaagacatg agaacctggc tcaagtatgc aagcctgtgc 4980

ggcaagagtg gcaggctggc tcttgctcat aaaactttag tgttgctcct gggagttgat 5040

ccgtctcggc aacttgacca tcctctgcca acagttcacc ctcaggtgac ctatgcctac 5100

atgaaaaaca tgtggaagag tgcccgcaag atcgatgcct tccagcacat gcagcatttt 5160

gtccagacca tgcagcaaca ggcccagcat gccatcgcta ctgaggacca gcagcataag 5220

caggaactgc acaagctcat ggcccgatgc ttcctgaaac ttggagagtg gcagctgaat 5280

ctacagggca tcaatgagag cacaatcccc aaagtgctgc agtactacag cgccgccaca 5340

gagcacgacc gcagctggta caaggcctgg catgcgtggg cagtgatgaa cttcgaagct 5400

gtgctacact acaaacatca gaaccaagcc cgcgatgaga agaagaaact gcgtcatgcc 5460

agcggggcca acatcaccaa cgccaccact gccgccacca cggccgccac tgccaccacc 5520

actgccagca ccgagggcag caacagtgag agcgaggccg agagcaccga gaacagcccc 5580

accccatcgc cgctgcagaa gaaggtcact gaggatctgt ccaaaaccct cctgatgtac 5640

acggtgcctg ccgtccaggg cttcttccgt tccatctcct tgtcacgagg caacaacctc 5700

caggatacac tcagagttct caccttatgg tttgattatg gtcactggcc agatgtcaat 5760

gaggccttag tggagggggt gaaagccatc cagattgata cctggctaca ggttatacct 5820

cagctcattg caagaattga tacgcccaga cccttggtgg gacgtctcat tcaccagctt 5880

ctcacagaca ttggtcggta ccacccccag gccctcatct acccactgac agtggcttct 5940

aagtctacca cgacagcccg gcacaatgca gccaacaaga ttctgaagaa catgtgtgag 6000

cacagcaaca ccctggtcca gcaggccatg atggtgagcg aggagctgat ccgagtggcc 6060

atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg 6120

gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg 6180

ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag 6240

gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cacccaagcc 6300

tgggacctct attatcatgt gttccgacga atctcaaagc agctgcctca gctcacatcc 6360

ttagagctgc aatatgtttc cccaaaactt ctgatgtgcc gggaccttga attggctgtg 6420

ccaggaacat atgaccccaa ccagccaatc attcgcattc agtccatagc accgtctttg 6480

caagtcatca catccaagca gaggccccgg aaattgacac ttatgggcag caacggacat 6540

gagtttgttt tccttctaaa aggccatgaa gatctgcgcc aggatgagcg tgtgatgcag 6600

ctcttcggcc tggttaacac ccttctggcc aatgacccaa catctcttcg gaaaaacctc 6660

agcatccaga gatacgctgt catcccttta tcgaccaact cgggcctcat tggctgggtt 6720

ccccactgtg acacactgca cgccctcatc cgggactaca gggagaagaa gaagatcctt 6780

ctcaacatcg agcatcgcat catgttgcgg atggctccgg actatgacca cttgactctg 6840

atgcagaagg tggaggtgtt tgagcatgcc gtcaataata cagctgggga cgacctggcc 6900

aagctgctgt ggctgaaaag ccccagctcc gaggtgtggt ttgaccgaag aaccaattat 6960

acccgttctt tagcggtcat gtcaatggtt gggtatattt taggcctggg agatagacac 7020

ccatccaacc tgatgctgga ccgtctgagt gggaagatcc tgcacattga ctttggggac 7080

tgctttgagg ttgctatgac ccgagagaag tttccagaga agattccatt tagactaaca 7140

agaatgttga ccaatgctat ggaggttaca ggcctggatg gcaactacag aatcacatgc 7200

cacacagtga tggaggtgct gcgagagcac aaggacagtg tcatggccgt gctggaagcc 7260

tttgtctatg accccttgct gaactggagg ctgatggaca caaataccaa aggcaacaag 7320

cgatcccgaa cgaggacgga ttcctactct gctggccagt cagtcgaaat tttggacggt 7380

gtggaacttg gagagccagc ccataagaaa acggggacca cagtgccaga atctattcat 7440

tctttcattg gagacggttt ggtgaaacca gaggccctaa ataagaaagc tatccagatt 7500

attaacaggg ttcgagataa gctcactggt cgggacttct ctcatgatga cactttggat 7560

gttccaacgc aagttgagct gctcatcaaa caagcgacat cccatgaaaa cctctgccag 7620

tgctatattg gctggtgccc tttctggtaa 7650

<210> 67

<211> 1147

<212> PRT

<213> Chile person

<400> 67

Met Asp Glu Pro Pro Phe Ser Glu Ala Ala Leu Glu Gln Ala Leu Gly

1 5 10 15

Glu Pro Cys Asp Leu Asp Ala Ala Leu Leu Thr Asp Ile Glu Asp Met

20 25 30

Leu Gln Leu Ile Asn Asn Gln Asp Ser Asp Phe Pro Gly Leu Phe Asp

35 40 45

Pro Pro Tyr Ala Gly Ser Gly Ala Gly Gly Thr Asp Pro Ala Ser Pro

50 55 60

Asp Thr Ser Ser Pro Gly Ser Leu Ser Pro Pro Pro Ala Thr Leu Ser

65 70 75 80

Ser Ser Leu Glu Ala Phe Leu Ser Gly Pro Gln Ala Ala Pro Ser Pro

85 90 95

Leu Ser Pro Pro Gln Pro Ala Pro Thr Pro Leu Lys Met Tyr Pro Ser

100 105 110

Met Pro Ala Phe Ser Pro Gly Pro Gly Ile Lys Glu Glu Ser Val Pro

115 120 125

Leu Ser Ile Leu Gln Thr Pro Thr Pro Gln Pro Leu Pro Gly Ala Leu

130 135 140

Leu Pro Gln Ser Phe Pro Ala Pro Ala Pro Pro Gln Phe Ser Ser Thr

145 150 155 160

Pro Val Leu Gly Tyr Pro Ser Pro Pro Gly Gly Phe Ser Thr Gly Ser

165 170 175

Pro Pro Gly Asn Thr Gln Gln Pro Leu Pro Gly Leu Pro Leu Ala Ser

180 185 190

Pro Pro Gly Val Pro Pro Val Ser Leu His Thr Gln Val Gln Ser Val

195 200 205

Val Pro Gln Gln Leu Leu Thr Val Thr Ala Ala Pro Thr Ala Ala Pro

210 215 220

Val Thr Thr Thr Val Thr Ser Gln Ile Gln Gln Val Pro Val Leu Leu

225 230 235 240

Gln Pro His Phe Ile Lys Ala Asp Ser Leu Leu Leu Thr Ala Met Lys

245 250 255

Thr Asp Gly Ala Thr Val Lys Ala Ala Gly Leu Ser Pro Leu Val Ser

260 265 270

Gly Thr Thr Val Gln Thr Gly Pro Leu Pro Thr Leu Val Ser Gly Gly

275 280 285

Thr Ile Leu Ala Thr Val Pro Leu Val Val Asp Ala Glu Lys Leu Pro

290 295 300

Ile Asn Arg Leu Ala Ala Gly Ser Lys Ala Pro Ala Ser Ala Gln Ser

305 310 315 320

Arg Gly Glu Lys Arg Thr Ala His Asn Ala Ile Glu Lys Arg Tyr Arg

325 330 335

Ser Ser Ile Asn Asp Lys Ile Ile Glu Leu Lys Asp Leu Val Val Gly

340 345 350

Thr Glu Ala Lys Leu Asn Lys Ser Ala Val Leu Arg Lys Ala Ile Asp

355 360 365

Tyr Ile Arg Phe Leu Gln His Ser Asn Gln Lys Leu Lys Gln Glu Asn

370 375 380

Leu Ser Leu Arg Thr Ala Val His Lys Ser Lys Ser Leu Lys Asp Leu

385 390 395 400

Val Ser Ala Cys Gly Ser Gly Gly Asn Thr Asp Val Leu Met Glu Gly

405 410 415

Val Lys Thr Glu Val Glu Asp Thr Leu Thr Pro Pro Pro Ser Asp Ala

420 425 430

Gly Ser Pro Phe Gln Ser Ser Pro Leu Ser Leu Gly Ser Arg Gly Ser

435 440 445

Gly Ser Gly Gly Ser Gly Ser Asp Ser Glu Pro Asp Ser Pro Val Phe

450 455 460

Glu Asp Ser Lys Ala Lys Pro Glu Gln Arg Pro Ser Leu His Ser Arg

465 470 475 480

Gly Met Leu Asp Arg Ser Arg Leu Ala Leu Cys Thr Leu Val Phe Leu

485 490 495

Cys Leu Ser Cys Asn Pro Leu Ala Ser Leu Leu Gly Ala Arg Gly Leu

500 505 510

Pro Ser Pro Ser Asp Thr Thr Ser Val Tyr His Ser Pro Gly Arg Asn

515 520 525

Val Leu Gly Thr Glu Ser Arg Asp Gly Pro Gly Trp Ala Gln Trp Leu

530 535 540

Leu Pro Pro Val Val Trp Leu Leu Asn Gly Leu Leu Val Leu Val Ser

545 550 555 560

Leu Val Leu Leu Phe Val Tyr Gly Glu Pro Val Thr Arg Pro His Ser

565 570 575

Gly Pro Ala Val Tyr Phe Trp Arg His Arg Lys Gln Ala Asp Leu Asp

580 585 590

Leu Ala Arg Gly Asp Phe Ala Gln Ala Ala Gln Gln Leu Trp Leu Ala

595 600 605

Leu Arg Ala Leu Gly Arg Pro Leu Pro Thr Ser His Leu Asp Leu Ala

610 615 620

Cys Ser Leu Leu Trp Asn Leu Ile Arg His Leu Leu Gln Arg Leu Trp

625 630 635 640

Val Gly Arg Trp Leu Ala Gly Arg Ala Gly Gly Leu Gln Gln Asp Cys

645 650 655

Ala Leu Arg Val Asp Ala Ser Ala Ser Ala Arg Asp Ala Ala Leu Val

660 665 670

Tyr His Lys Leu His Gln Leu His Thr Met Gly Lys His Thr Gly Gly

675 680 685

His Leu Thr Ala Thr Asn Leu Ala Leu Ser Ala Leu Asn Leu Ala Glu

690 695 700

Cys Ala Gly Asp Ala Val Ser Val Ala Thr Leu Ala Glu Ile Tyr Val

705 710 715 720

Ala Ala Ala Leu Arg Val Lys Thr Ser Leu Pro Arg Ala Leu His Phe

725 730 735

Leu Thr Arg Phe Phe Leu Ser Ser Ala Arg Gln Ala Cys Leu Ala Gln

740 745 750

Ser Gly Ser Val Pro Pro Ala Met Gln Trp Leu Cys His Pro Val Gly

755 760 765

His Arg Phe Phe Val Asp Gly Asp Trp Ser Val Leu Ser Thr Pro Trp

770 775 780

Glu Ser Leu Tyr Ser Leu Ala Gly Asn Pro Val Asp Pro Leu Ala Gln

785 790 795 800

Val Thr Gln Leu Phe Arg Glu His Leu Leu Glu Arg Ala Leu Asn Cys

805 810 815

Val Thr Gln Pro Asn Pro Ser Pro Gly Ser Ala Asp Gly Asp Lys Glu

820 825 830

Phe Ser Asp Ala Leu Gly Tyr Leu Gln Leu Leu Asn Ser Cys Ser Asp

835 840 845

Ala Ala Gly Ala Pro Ala Tyr Ser Phe Ser Ile Ser Ser Ser Met Ala

850 855 860

Thr Thr Thr Gly Val Asp Pro Val Ala Lys Trp Trp Ala Ser Leu Thr

865 870 875 880

Ala Val Val Ile His Trp Leu Arg Arg Asp Glu Glu Ala Ala Glu Arg

885 890 895

Leu Cys Pro Leu Val Glu His Leu Pro Arg Val Leu Gln Glu Ser Glu

900 905 910

Arg Pro Leu Pro Arg Ala Ala Leu His Ser Phe Lys Ala Ala Arg Ala

915 920 925

Leu Leu Gly Cys Ala Lys Ala Glu Ser Gly Pro Ala Ser Leu Thr Ile

930 935 940

Cys Glu Lys Ala Ser Gly Tyr Leu Gln Asp Ser Leu Ala Thr Thr Pro

945 950 955 960

Ala Ser Ser Ser Ile Asp Lys Ala Val Gln Leu Phe Leu Cys Asp Leu

965 970 975

Leu Leu Val Val Arg Thr Ser Leu Trp Arg Gln Gln Gln Pro Pro Ala

980 985 990

Pro Ala Pro Ala Ala Gln Gly Thr Ser Ser Arg Pro Gln Ala Ser Ala

995 1000 1005

Leu Glu Leu Arg Gly Phe Gln Arg Asp Leu Ser Ser Leu Arg Arg

1010 1015 1020

Leu Ala Gln Ser Phe Arg Pro Ala Met Arg Arg Val Phe Leu His

1025 1030 1035

Glu Ala Thr Ala Arg Leu Met Ala Gly Ala Ser Pro Thr Arg Thr

1040 1045 1050

His Gln Leu Leu Asp Arg Ser Leu Arg Arg Arg Ala Gly Pro Gly

1055 1060 1065

Gly Lys Gly Gly Ala Val Ala Glu Leu Glu Pro Arg Pro Thr Arg

1070 1075 1080

Arg Glu His Ala Glu Ala Leu Leu Leu Ala Ser Cys Tyr Leu Pro

1085 1090 1095

Pro Gly Phe Leu Ser Ala Pro Gly Gln Arg Val Gly Met Leu Ala

1100 1105 1110

Glu Ala Ala Arg Thr Leu Glu Lys Leu Gly Asp Arg Arg Leu Leu

1115 1120 1125

His Asp Cys Gln Gln Met Leu Met Arg Leu Gly Gly Gly Thr Thr

1130 1135 1140

Val Thr Ser Ser

1145

<210> 68

<211> 3444

<212> DNA

<213> Chile person

<400> 68

atggacgagc cacccttcag cgaggcggct ttggagcagg cgctgggcga gccgtgcgat 60

ctggacgcgg cgctgctgac cgacatcgaa gacatgcttc agcttatcaa caaccaagac 120

agtgacttcc ctggcctatt tgacccaccc tatgctggga gtggggcagg gggcacagac 180

cctgccagcc ccgataccag ctccccaggc agcttgtctc cacctcctgc cacattgagc 240

tcctctcttg aagccttcct gagcgggccg caggcagcgc cctcacccct gtcccctccc 300

cagcctgcac ccactccatt gaagatgtac ccgtccatgc ccgctttctc ccctgggcct 360

ggtatcaagg aagagtcagt gccactgagc atcctgcaga cccccacccc acagcccctg 420

ccaggggccc tcctgccaca gagcttccca gccccagccc caccgcagtt cagctccacc 480

cctgtgttag gctaccccag ccctccggga ggcttctcta caggaagccc tcccgggaac 540

acccagcagc cgctgcctgg cctgccactg gcttccccgc caggggtccc gcccgtctcc 600

ttgcacaccc aggtccagag tgtggtcccc cagcagctac tgacagtcac agctgccccc 660

acggcagccc ctgtaacgac cactgtgacc tcgcagatcc agcaggtccc ggtcctgctg 720

cagccccact tcatcaaggc agactcgctg cttctgacag ccatgaagac agacggagcc 780

actgtgaagg cggcaggtct cagtcccctg gtctctggca ccactgtgca gacagggcct 840

ttgccgaccc tggtgagtgg cggaaccatc ttggcaacag tcccactggt cgtagatgcg 900

gagaagctgc ctatcaaccg gctcgcagct ggcagcaagg ccccggcctc tgcccagagc 960

cgtggagaga agcgcacagc ccacaacgcc attgagaagc gctaccgctc ctccatcaat 1020

gacaaaatca ttgagctcaa ggatctggtg gtgggcactg aggcaaagct gaataaatct 1080

gctgtcttgc gcaaggccat cgactacatt cgctttctgc aacacagcaa ccagaaactc 1140

aagcaggaga acctaagtct gcgcactgct gtccacaaaa gcaaatctct gaaggatctg 1200

gtgtcggcct gtggcagtgg agggaacaca gacgtgctca tggagggcgt gaagactgag 1260

gtggaggaca cactgacccc acccccctcg gatgctggct cacctttcca gagcagcccc 1320

ttgtcccttg gcagcagggg cagtggcagc ggtggcagtg gcagtgactc ggagcctgac 1380

agcccagtct ttgaggacag caaggcaaag ccagagcagc ggccgtctct gcacagccgg 1440

ggcatgctgg accgctcccg cctggccctg tgcacgctcg tcttcctctg cctgtcctgc 1500

aaccccttgg cctccttgct gggggcccgg gggcttccca gcccctcaga taccaccagc 1560

gtctaccata gccctgggcg caacgtgctg ggcaccgaga gcagagatgg ccctggctgg 1620

gcccagtggc tgctgccccc agtggtctgg ctgctcaatg ggctgttggt gctcgtctcc 1680

ttggtgcttc tctttgtcta cggtgagcca gtcacacggc cccactcagg ccccgccgtg 1740

tacttctgga ggcatcgcaa gcaggctgac ctggacctgg cccggggaga ctttgcccag 1800

gctgcccagc agctgtggct ggccctgcgg gcactgggcc ggcccctgcc cacctcccac 1860

ctggacctgg cttgtagcct cctctggaac ctcatccgtc acctgctgca gcgtctctgg 1920

gtgggccgct ggctggcagg ccgggcaggg ggcctgcagc aggactgtgc tctgcgagtg 1980

gatgctagcg ccagcgcccg agacgcagcc ctggtctacc ataagctgca ccagctgcac 2040

accatgggga agcacacagg cgggcacctc actgccacca acctggcgct gagtgccctg 2100

aacctggcag agtgtgcagg ggatgccgtg tctgtggcga cgctggccga gatctatgtg 2160

gcggctgcat tgagagtgaa gaccagtctc ccacgggcct tgcattttct gacacgcttc 2220

ttcctgagca gtgcccgcca ggcctgcctg gcacagagtg gctcagtgcc tcctgccatg 2280

cagtggctct gccaccccgt gggccaccgt ttcttcgtgg atggggactg gtccgtgctc 2340

agtaccccat gggagagcct gtacagcttg gccgggaacc cagtggaccc cctggcccag 2400

gtgactcagc tattccggga acatctctta gagcgagcac tgaactgtgt gacccagccc 2460

aaccccagcc ctgggtcagc tgatggggac aaggaattct cggatgccct cgggtacctg 2520

cagctgctga acagctgttc tgatgctgcg ggggctcctg cctacagctt ctccatcagt 2580

tccagcatgg ccaccaccac cggcgtagac ccggtggcca agtggtgggc ctctctgaca 2640

gctgtggtga tccactggct gcggcgggat gaggaggcgg ctgagcggct gtgcccgctg 2700

gtggagcacc tgccccgggt gctgcaggag tctgagagac ccctgcccag ggcagctctg 2760

cactccttca aggctgcccg ggccctgctg ggctgtgcca aggcagagtc tggtccagcc 2820

agcctgacca tctgtgagaa ggccagtggg tacctgcagg acagcctggc taccacacca 2880

gccagcagct ccattgacaa ggccgtgcag ctgttcctgt gtgacctgct tcttgtggtg 2940

cgcaccagcc tgtggcggca gcagcagccc ccggccccgg ccccagcagc ccagggcacc 3000

agcagcaggc cccaggcttc cgcccttgag ctgcgtggct tccaacggga cctgagcagc 3060

ctgaggcggc tggcacagag cttccggccc gccatgcgga gggtgttcct acatgaggcc 3120

acggcccggc tgatggcggg ggccagcccc acacggacac accagctcct cgaccgcagt 3180

ctgaggcggc gggcaggccc cggtggcaaa ggaggcgcgg tggcggagct ggagccgcgg 3240

cccacgcggc gggagcacgc ggaggccttg ctgctggcct cctgctacct gccccccggc 3300

ttcctgtcgg cgcccgggca gcgcgtgggc atgctggctg aggcggcgcg cacactcgag 3360

aagcttggcg atcgccggct gctgcacgac tgtcagcaga tgctcatgcg cctgggcggt 3420

gggaccactg tcacttccag ctag 3444

<210> 69

<211> 138

<212> PRT

<213> Chile person

<400> 69

Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn

1 5 10 15

Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile

20 25 30

Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln

35 40 45

Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln

50 55 60

Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys

65 70 75 80

Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr

85 90 95

Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu

100 105 110

Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys

115 120 125

Arg Lys Arg Ser Gln Met Leu Phe Arg Gly

130 135

<210> 70

<211> 414

<212> DNA

<213> Chile person

<400> 70

caggacccat atgtaaaaga agcagaaaac cttaagaaat attttaatgc aggtcattca 60

gatgtagcgg ataatggaac tcttttctta ggcattttga agaattggaa agaggagagt 120

gacagaaaaa taatgcagag ccaaattgtc tccttttact tcaaactttt taaaaacttt 180

aaagatgacc agagcatcca aaagagtgtg gagaccatca aggaagacat gaatgtcaag 240

tttttcaata gcaacaaaaa gaaacgagat gacttcgaaa agctgactaa ttattcggta 300

actgacttga atgtccaacg caaagcaata catgaactca tccaagtgat ggctgaactg 360

tcgccagcag ctaaaacagg gaagcgaaaa aggagtcaga tgctgtttcg aggt 414

<210> 71

<211> 227

<212> PRT

<213> Chile person

<400> 71

Ile Ile Gly Gly His Glu Ala Lys Pro His Ser Arg Pro Tyr Met Ala

1 5 10 15

Tyr Leu Met Ile Trp Asp Gln Lys Ser Leu Lys Arg Cys Gly Gly Phe

20 25 30

Leu Ile Arg Asp Asp Phe Val Leu Thr Ala Ala His Cys Trp Gly Ser

35 40 45

Ser Ile Asn Val Thr Leu Gly Ala His Asn Ile Lys Glu Gln Glu Pro

50 55 60

Thr Gln Gln Phe Ile Pro Val Lys Arg Pro Ile Pro His Pro Ala Tyr

65 70 75 80

Asn Pro Lys Asn Phe Ser Asn Asp Ile Met Leu Leu Gln Leu Glu Arg

85 90 95

Lys Ala Lys Arg Thr Arg Ala Val Gln Pro Leu Arg Leu Pro Ser Asn

100 105 110

Lys Ala Gln Val Lys Pro Gly Gln Thr Cys Ser Val Ala Gly Trp Gly

115 120 125

Gln Thr Ala Pro Leu Gly Lys His Ser His Thr Leu Gln Glu Val Lys

130 135 140

Met Thr Val Gln Glu Asp Arg Lys Cys Glu Ser Asp Leu Arg His Tyr

145 150 155 160

Tyr Asp Ser Thr Ile Glu Leu Cys Val Gly Asp Pro Glu Ile Lys Lys

165 170 175

Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Lys Val

180 185 190

Ala Gln Gly Ile Val Ser Tyr Gly Arg Asn Asn Gly Met Pro Pro Arg

195 200 205

Ala Cys Thr Lys Val Ser Ser Phe Val His Trp Ile Lys Lys Thr Met

210 215 220

Lys Arg Tyr

225

<210> 72

<211> 681

<212> DNA

<213> Chile person

<400> 72

atcatcgggg gacatgaggc caagccccac tcccgcccct acatggctta tcttatgatc 60

tgggatcaga agtctctgaa gaggtgcggt ggcttcctga tacgagacga cttcgtgctg 120

acagctgctc actgttgggg aagctccata aatgtcacct tgggggccca caatatcaaa 180

gaacaggagc cgacccagca gtttatccct gtgaaaagac ccatccccca tccagcctat 240

aatcctaaga acttctccaa cgacatcatg ctactgcagc tggagagaaa ggccaagcgg 300

accagagctg tgcagcccct caggctacct agcaacaagg cccaggtgaa gccagggcag 360

acatgcagtg tggccggctg ggggcagacg gcccccctgg gaaaacactc acacacacta 420

caagaggtga agatgacagt gcaggaagat cgaaagtgcg aatctgactt acgccattat 480

tacgacagta ccattgagtt gtgcgtgggg gacccagaga ttaaaaagac ttcctttaag 540

ggggactctg gaggccctct tgtgtgtaac aaggtggccc agggcattgt ctcctatgga 600

cgaaacaatg gcatgcctcc acgagcctgc accaaagtct caagctttgt acactggata 660

aagaaaacca tgaaacgcta c 681

<210> 73

<211> 268

<212> PRT

<213> Chile person

<400> 73

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly Leu Leu Gly Ser

145 150 155 160

Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys Ser Arg Ala Ala

165 170 175

Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro Leu Lys Glu Asp

180 185 190

Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly Glu Leu Asp Phe

195 200 205

Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro Cys Val Pro Glu

210 215 220

Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly Met Gly Thr Ser

225 230 235 240

Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg Ser Ala Gln Pro

245 250 255

Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

260 265

<210> 74

<211> 272

<212> PRT

<213> Chile person

<400> 74

Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu

20 25 30

Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln

35 40 45

Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg

50 55 60

Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys

85 90 95

Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val

100 105 110

Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro

115 120 125

Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys

130 135 140

Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys

145 150 155 160

Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr

165 170 175

Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr

180 185 190

Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile

195 200 205

Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His Leu Val

210 215 220

Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr Phe Ile

225 230 235 240

Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys Gly Ile

245 250 255

Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu Glu Thr

260 265 270

<210> 75

<211> 223

<212> PRT

<213> Chile person

<400> 75

Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys

1 5 10 15

Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln

20 25 30

Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys

35 40 45

Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val

50 55 60

Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn

65 70 75 80

Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr

85 90 95

Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu

100 105 110

His Gly Ala Arg Phe Gln Ile Pro Leu Leu Gly Ala Met Ala Ala Thr

115 120 125

Leu Val Val Ile Cys Thr Ala Val Ile Val Val Val Ala Leu Thr Arg

130 135 140

Lys Lys Lys Ala Leu Arg Ile His Ser Val Glu Gly Asp Leu Arg Arg

145 150 155 160

Lys Ser Ala Gly Gln Glu Glu Trp Ser Pro Ser Ala Pro Ser Pro Pro

165 170 175

Gly Ser Cys Val Gln Ala Glu Ala Ala Pro Ala Gly Leu Cys Gly Glu

180 185 190

Gln Arg Gly Glu Asp Cys Ala Glu Leu His Asp Tyr Phe Asn Val Leu

195 200 205

Ser Tyr Arg Ser Leu Gly Asn Cys Ser Phe Phe Thr Glu Thr Gly

210 215 220

<210> 76

<211> 311

<212> PRT

<213> Chile person

<400> 76

Asn Ala Asp Gly Leu Lys Glu Pro Leu Ser Phe His Val Thr Trp Ile

1 5 10 15

Ala Ser Phe Tyr Asn His Ser Trp Lys Gln Asn Leu Val Ser Gly Trp

20 25 30

Leu Ser Asp Leu Gln Thr His Thr Trp Asp Ser Asn Ser Ser Thr Ile

35 40 45

Val Phe Leu Cys Pro Trp Ser Arg Gly Asn Phe Ser Asn Glu Glu Trp

50 55 60

Lys Glu Leu Glu Thr Leu Phe Arg Ile Arg Thr Ile Arg Ser Phe Glu

65 70 75 80

Gly Ile Arg Arg Tyr Ala His Glu Leu Gln Phe Glu Tyr Pro Phe Glu

85 90 95

Ile Gln Val Thr Gly Gly Cys Glu Leu His Ser Gly Lys Val Ser Gly

100 105 110

Ser Phe Leu Gln Leu Ala Tyr Gln Gly Ser Asp Phe Val Ser Phe Gln

115 120 125

Asn Asn Ser Trp Leu Pro Tyr Pro Val Ala Gly Asn Met Ala Lys His

130 135 140

Phe Cys Lys Val Leu Asn Gln Asn Gln His Glu Asn Asp Ile Thr His

145 150 155 160

Asn Leu Leu Ser Asp Thr Cys Pro Arg Phe Ile Leu Gly Leu Leu Asp

165 170 175

Ala Gly Lys Ala His Leu Gln Arg Gln Val Lys Pro Glu Ala Trp Leu

180 185 190

Ser His Gly Pro Ser Pro Gly Pro Gly His Leu Gln Leu Val Cys His

195 200 205

Val Ser Gly Phe Tyr Pro Lys Pro Val Trp Val Met Trp Met Arg Gly

210 215 220

Glu Gln Glu Gln Gln Gly Thr Gln Arg Gly Asp Ile Leu Pro Ser Ala

225 230 235 240

Asp Gly Thr Trp Tyr Leu Arg Ala Thr Leu Glu Val Ala Ala Gly Glu

245 250 255

Ala Ala Asp Leu Ser Cys Arg Val Lys His Ser Ser Leu Glu Gly Gln

260 265 270

Asp Ile Val Leu Tyr Trp Glu His His Ser Ser Val Gly Phe Ile Ile

275 280 285

Leu Ala Val Ile Val Pro Leu Leu Leu Leu Ile Gly Leu Ala Leu Trp

290 295 300

Phe Arg Lys Arg Cys Phe Cys

305 310

<210> 77

<211> 279

<212> PRT

<213> Chile person

<400> 77

Ser Glu Val Glu Tyr Arg Ala Glu Val Gly Gln Asn Ala Tyr Leu Pro

1 5 10 15

Cys Phe Tyr Thr Pro Ala Ala Pro Gly Asn Leu Val Pro Val Cys Trp

20 25 30

Gly Lys Gly Ala Cys Pro Val Phe Glu Cys Gly Asn Val Val Leu Arg

35 40 45

Thr Asp Glu Arg Asp Val Asn Tyr Trp Thr Ser Arg Tyr Trp Leu Asn

50 55 60

Gly Asp Phe Arg Lys Gly Asp Val Ser Leu Thr Ile Glu Asn Val Thr

65 70 75 80

Leu Ala Asp Ser Gly Ile Tyr Cys Cys Arg Ile Gln Ile Pro Gly Ile

85 90 95

Met Asn Asp Glu Lys Phe Asn Leu Lys Leu Val Ile Lys Pro Ala Lys

100 105 110

Val Thr Pro Ala Pro Thr Arg Gln Arg Asp Phe Thr Ala Ala Phe Pro

115 120 125

Arg Met Leu Thr Thr Arg Gly His Gly Pro Ala Glu Thr Gln Thr Leu

130 135 140

Gly Ser Leu Pro Asp Ile Asn Leu Thr Gln Ile Ser Thr Leu Ala Asn

145 150 155 160

Glu Leu Arg Asp Ser Arg Leu Ala Asn Asp Leu Arg Asp Ser Gly Ala

165 170 175

Thr Ile Arg Ile Gly Ile Tyr Ile Gly Ala Gly Ile Cys Ala Gly Leu

180 185 190

Ala Leu Ala Leu Ile Phe Gly Ala Leu Ile Phe Lys Trp Tyr Ser His

195 200 205

Ser Lys Glu Lys Ile Gln Asn Leu Ser Leu Ile Ser Leu Ala Asn Leu

210 215 220

Pro Pro Ser Gly Leu Ala Asn Ala Val Ala Glu Gly Ile Arg Ser Glu

225 230 235 240

Glu Asn Ile Tyr Thr Ile Glu Glu Asn Val Tyr Glu Val Glu Glu Pro

245 250 255

Asn Glu Tyr Tyr Cys Tyr Val Ser Ser Arg Gln Gln Pro Ser Gln Pro

260 265 270

Leu Gly Cys Arg Phe Ala Met

275

<210> 78

<211> 133

<212> PRT

<213> Chile person

<400> 78

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 79

<211> 152

<212> PRT

<213> Chile person

<400> 79

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His

145 150

<210> 80

<211> 197

<212> PRT

<213> Chile person

<400> 80

Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu

1 5 10 15

His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys

20 25 30

Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp

35 40 45

His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu

50 55 60

Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr

65 70 75 80

Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe

85 90 95

Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr

100 105 110

Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys

115 120 125

Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu

130 135 140

Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser

145 150 155 160

Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu

165 170 175

Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser

180 185 190

Tyr Leu Asn Ala Ser

195

<210> 81

<211> 306

<212> PRT

<213> Chile person

<400> 81

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

Cys Ser

305

<210> 82

<211> 114

<212> PRT

<213> Chile person

<400> 82

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 83

<211> 133

<212> PRT

<213> Chile person

<400> 83

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser

130

<210> 84

<211> 270

<212> PRT

<213> Chile person

<400> 84

Met Lys Pro Lys Met Lys Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys

1 5 10 15

Trp Lys Asn Thr Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser

20 25 30

Gln Gln Lys Ala Lys Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg

35 40 45

Ser Gly Leu Met Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr

50 55 60

Thr Lys Arg Pro Ser Leu Lys Thr Gly Arg Lys His Lys Arg His Leu

65 70 75 80

Val Leu Ala Ala Cys Gln Gln Gln Ser Thr Val Glu Cys Phe Ala Phe

85 90 95

Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp Ser Ser

100 105 110

Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser Thr

115 120 125

Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser Tyr Glu

130 135 140

Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys Val Leu

145 150 155 160

Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly Asp Gly

165 170 175

Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe

180 185 190

Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys Cys

195 200 205

Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met His

210 215 220

Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe Ile

225 230 235 240

Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser Glu

245 250 255

Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr

260 265 270

<210> 85

<211> 696

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 85

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

145 150 155 160

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

165 170 175

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

180 185 190

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

210 215 220

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

225 230 235 240

Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys

245 250 255

Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn

260 265 270

Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser

275 280 285

Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile

290 295 300

Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro

305 310 315 320

Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu

325 330 335

Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro

340 345 350

Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val

355 360 365

Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu

370 375 380

Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys

385 390 395 400

Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe

405 410 415

Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala

420 425 430

Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val

435 440 445

Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Val Gln Leu Gln

450 455 460

Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser

465 470 475 480

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Ile Gln Trp Val

485 490 495

Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Ser Ile Asn Pro

500 505 510

Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr

515 520 525

Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala Tyr Met Glu Phe Ser Ser

530 535 540

Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys Ala Arg Trp Gly Asp

545 550 555 560

Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

565 570 575

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu

580 585 590

Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Arg Val

595 600 605

Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Phe His

610 615 620

Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Cys Ile Tyr Ser

625 630 635 640

Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser Gly Ser Gly

645 650 655

Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala

660 665 670

Ala Thr Tyr Phe Cys His Gln Tyr His Arg Ser Pro Thr Phe Gly Gly

675 680 685

Gly Thr Lys Leu Glu Thr Lys Arg

690 695

<210> 86

<211> 2088

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 86

cagatcgtgc tgacccaaag ccccgccatc atgagcgcta gccccggtga gaaggtgacc 60

atgacatgct ccgcttccag ctccgtgtcc tacatgaact ggtatcagca gaaaagcgga 120

accagcccca aaaggtggat ctacgacacc agcaagctgg cctccggagt gcccgctcat 180

ttccggggct ctggatccgg caccagctac tctttaacca tttccggcat ggaagctgaa 240

gacgctgcca cctactattg ccagcaatgg agcagcaacc ccttcacatt cggatctggc 300

accaagctcg aaatcaatcg tggaggaggt ggcagcggcg gcggtggatc cggcggagga 360

ggaagccaag ttcaactcca gcagagcggc gctgaactgg cccggcccgg cgcctccgtc 420

aagatgagct gcaaggcttc cggctataca tttactcgtt acacaatgca ttgggtcaag 480

cagaggcccg gtcaaggttt agagtggatc ggatatatca acccttcccg gggctacacc 540

aactataacc aaaagttcaa ggataaagcc actttaacca ctgacaagag ctcctccacc 600

gcctacatgc agctgtcctc tttaaccagc gaggactccg ctgtttacta ctgcgctagg 660

tattacgacg accactactg tttagactat tggggacaag gtaccacttt aaccgtcagc 720

agctccggca ccaccaatac cgtggccgct tataacctca catggaagag caccaacttc 780

aagacaattc tggaatggga acccaagccc gtcaatcaag tttacaccgt gcagatctcc 840

accaaatccg gagactggaa gagcaagtgc ttctacacaa cagacaccga gtgtgattta 900

accgacgaaa tcgtcaagga cgtcaagcaa acctatctgg ctcgggtctt ttcctacccc 960

gctggcaatg tcgagtccac cggctccgct ggcgagcctc tctacgagaa ttcccccgaa 1020

ttcacccctt atttagagac caatttaggc cagcctacca tccagagctt cgagcaagtt 1080

ggcaccaagg tgaacgtcac cgtcgaggat gaaaggactt tagtgcggcg gaataacaca 1140

tttttatccc tccgggatgt gttcggcaaa gacctcatct acacactgta ctattggaag 1200

tccagctcct ccggcaaaaa gaccgctaag accaacacca acgagttttt aattgacgtg 1260

gacaaaggcg agaactactg cttcagcgtg caagccgtga tcccttctcg taccgtcaac 1320

cggaagagca cagattcccc cgttgagtgc atgggccaag aaaagggcga gttccgggag 1380

gtccagctgc agcagagcgg acccgaactc gtgaaacccg gtgcttccgt gaaaatgtct 1440

tgtaaggcca gcggatacac cttcacctcc tatgtgatcc agtgggtcaa acagaagccc 1500

ggacaaggtc tcgagtggat cggcagcatc aacccttaca acgactatac caaatacaac 1560

gagaagttta agggaaaggc tactttaacc tccgacaaaa gctccatcac agcctacatg 1620

gagttcagct ctttaacatc cgaggacagc gctctgtact attgcgcccg gtggggcgac 1680

ggcaattact ggggacgggg cacaacactg accgtgagca gcggaggcgg aggctccggc 1740

ggaggcggat ctggcggtgg cggctccgac atcgagatga cccagtcccc cgctatcatg 1800

tccgcctctt taggcgagcg ggtcacaatg acttgtacag cctcctccag cgtctcctcc 1860

tcctacttcc attggtacca acagaaaccc ggaagctccc ctaaactgtg catctacagc 1920

accagcaatc tcgccagcgg cgtgccccct aggttttccg gaagcggaag caccagctac 1980

tctttaacca tctcctccat ggaggctgag gatgccgcca cctacttttg tcaccagtac 2040

caccggtccc ccaccttcgg aggcggcacc aaactggaga caaagagg 2088

<210> 87

<211> 714

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 87

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser

20 25 30

Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser

35 40 45

Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp

50 55 60

Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg

65 70 75 80

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu

85 90 95

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro

100 105 110

Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

130 135 140

Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met

145 150 155 160

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

165 170 175

Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn

180 185 190

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala

195 200 205

Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser

210 215 220

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

225 230 235 240

Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

245 250 255

Val Ser Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr

260 265 270

Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro

275 280 285

Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp

290 295 300

Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp

305 310 315 320

Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser

325 330 335

Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu

340 345 350

Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly

355 360 365

Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val

370 375 380

Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu

385 390 395 400

Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr

405 410 415

Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn

420 425 430

Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val

435 440 445

Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser

450 455 460

Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Val Gln

465 470 475 480

Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys

485 490 495

Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Ile Gln

500 505 510

Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Ser Ile

515 520 525

Asn Pro Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys

530 535 540

Ala Thr Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala Tyr Met Glu Phe

545 550 555 560

Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys Ala Arg Trp

565 570 575

Gly Asp Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val Ser Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

595 600 605

Ile Glu Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu

610 615 620

Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr

625 630 635 640

Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Cys Ile

645 650 655

Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser Gly

660 665 670

Ser Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

675 680 685

Asp Ala Ala Thr Tyr Phe Cys His Gln Tyr His Arg Ser Pro Thr Phe

690 695 700

Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg

705 710

<210> 88

<211> 2142

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 88

atgaagtggg tgaccttcat cagcttatta tttttattca gctccgccta ttcccagatc 60

gtgctgaccc aaagccccgc catcatgagc gctagccccg gtgagaaggt gaccatgaca 120

tgctccgctt ccagctccgt gtcctacatg aactggtatc agcagaaaag cggaaccagc 180

cccaaaaggt ggatctacga caccagcaag ctggcctccg gagtgcccgc tcatttccgg 240

ggctctggat ccggcaccag ctactcttta accatttccg gcatggaagc tgaagacgct 300

gccacctact attgccagca atggagcagc aaccccttca cattcggatc tggcaccaag 360

ctcgaaatca atcgtggagg aggtggcagc ggcggcggtg gatccggcgg aggaggaagc 420

caagttcaac tccagcagag cggcgctgaa ctggcccggc ccggcgcctc cgtcaagatg 480

agctgcaagg cttccggcta tacatttact cgttacacaa tgcattgggt caagcagagg 540

cccggtcaag gtttagagtg gatcggatat atcaaccctt cccggggcta caccaactat 600

aaccaaaagt tcaaggataa agccacttta accactgaca agagctcctc caccgcctac 660

atgcagctgt cctctttaac cagcgaggac tccgctgttt actactgcgc taggtattac 720

gacgaccact actgtttaga ctattgggga caaggtacca ctttaaccgt cagcagctcc 780

ggcaccacca ataccgtggc cgcttataac ctcacatgga agagcaccaa cttcaagaca 840

attctggaat gggaacccaa gcccgtcaat caagtttaca ccgtgcagat ctccaccaaa 900

tccggagact ggaagagcaa gtgcttctac acaacagaca ccgagtgtga tttaaccgac 960

gaaatcgtca aggacgtcaa gcaaacctat ctggctcggg tcttttccta ccccgctggc 1020

aatgtcgagt ccaccggctc cgctggcgag cctctctacg agaattcccc cgaattcacc 1080

ccttatttag agaccaattt aggccagcct accatccaga gcttcgagca agttggcacc 1140

aaggtgaacg tcaccgtcga ggatgaaagg actttagtgc ggcggaataa cacattttta 1200

tccctccggg atgtgttcgg caaagacctc atctacacac tgtactattg gaagtccagc 1260

tcctccggca aaaagaccgc taagaccaac accaacgagt ttttaattga cgtggacaaa 1320

ggcgagaact actgcttcag cgtgcaagcc gtgatccctt ctcgtaccgt caaccggaag 1380

agcacagatt cccccgttga gtgcatgggc caagaaaagg gcgagttccg ggaggtccag 1440

ctgcagcaga gcggacccga actcgtgaaa cccggtgctt ccgtgaaaat gtcttgtaag 1500

gccagcggat acaccttcac ctcctatgtg atccagtggg tcaaacagaa gcccggacaa 1560

ggtctcgagt ggatcggcag catcaaccct tacaacgact ataccaaata caacgagaag 1620

tttaagggaa aggctacttt aacctccgac aaaagctcca tcacagccta catggagttc 1680

agctctttaa catccgagga cagcgctctg tactattgcg cccggtgggg cgacggcaat 1740

tactggggac ggggcacaac actgaccgtg agcagcggag gcggaggctc cggcggaggc 1800

ggatctggcg gtggcggctc cgacatcgag atgacccagt cccccgctat catgtccgcc 1860

tctttaggcg agcgggtcac aatgacttgt acagcctcct ccagcgtctc ctcctcctac 1920

ttccattggt accaacagaa acccggaagc tcccctaaac tgtgcatcta cagcaccagc 1980

aatctcgcca gcggcgtgcc ccctaggttt tccggaagcg gaagcaccag ctactcttta 2040

accatctcct ccatggaggc tgaggatgcc gccacctact tttgtcacca gtaccaccgg 2100

tcccccacct tcggaggcgg caccaaactg gagacaaaga gg 2142

<210> 89

<211> 696

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 89

Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val

20 25 30

Ile Gln Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly

35 40 45

Ser Ile Asn Pro Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys Phe Lys

50 55 60

Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala Tyr Met

65 70 75 80

Glu Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys Ala

85 90 95

Arg Trp Gly Asp Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Asp Ile Glu Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu

130 135 140

Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser

145 150 155 160

Ser Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu

165 170 175

Cys Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe

180 185 190

Ser Gly Ser Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

195 200 205

Ala Glu Asp Ala Ala Thr Tyr Phe Cys His Gln Tyr His Arg Ser Pro

210 215 220

Thr Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg Ser Gly Thr Thr

225 230 235 240

Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys

245 250 255

Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val

260 265 270

Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr

275 280 285

Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys

290 295 300

Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu

305 310 315 320

Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe

325 330 335

Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe

340 345 350

Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr

355 360 365

Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly

370 375 380

Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly

385 390 395 400

Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp

405 410 415

Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg

420 425 430

Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly Gln

435 440 445

Glu Lys Gly Glu Phe Arg Glu Gln Ile Val Leu Thr Gln Ser Pro Ala

450 455 460

Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala

465 470 475 480

Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr

485 490 495

Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val

500 505 510

Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr

515 520 525

Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

530 535 540

Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

545 550 555 560

Asn Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

565 570 575

Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

580 585 590

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg

595 600 605

Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp

610 615 620

Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys

625 630 635 640

Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala

645 650 655

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

660 665 670

Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln

675 680 685

Gly Thr Thr Leu Thr Val Ser Ser

690 695

<210> 90

<211> 2088

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 90

gtgcagctgc agcagtccgg acccgaactg gtcaagcccg gtgcctccgt gaaaatgtct 60

tgtaaggctt ctggctacac ctttacctcc tacgtcatcc aatgggtgaa gcagaagccc 120

ggtcaaggtc tcgagtggat cggcagcatc aatccctaca acgattacac caagtataac 180

gaaaagttta agggcaaggc cactctgaca agcgacaaga gctccattac cgcctacatg 240

gagttttcct ctttaacttc tgaggactcc gctttatact attgcgctcg ttggggcgat 300

ggcaattatt ggggccgggg aactacttta acagtgagct ccggcggcgg cggaagcgga 360

ggtggaggat ctggcggtgg aggcagcgac atcgagatga cacagtcccc cgctatcatg 420

agcgcctctt taggagaacg tgtgaccatg acttgtacag cttcctccag cgtgagcagc 480

tcctatttcc actggtacca gcagaaaccc ggctcctccc ctaaactgtg tatctactcc 540

acaagcaatt tagctagcgg cgtgcctcct cgttttagcg gctccggcag cacctcttac 600

tctttaacca ttagctctat ggaggccgaa gatgccgcca catacttttg ccatcagtac 660

caccggtccc ctacctttgg cggaggcaca aagctggaga ccaagcggag cggcaccacc 720

aacacagtgg ccgcctacaa tctgacttgg aaatccacca acttcaagac catcctcgag 780

tgggagccca agcccgttaa tcaagtttat accgtgcaga tttccaccaa gagcggcgac 840

tggaaatcca agtgcttcta taccacagac accgagtgcg atctcaccga cgagatcgtc 900

aaagacgtga agcagacata tttagctagg gtgttctcct accccgctgg aaacgtggag 960

agcaccggat ccgctggaga gcctttatac gagaactccc ccgaattcac cccctatctg 1020

gaaaccaatt taggccagcc caccatccag agcttcgaac aagttggcac aaaggtgaac 1080

gtcaccgtcg aagatgagag gactttagtg cggaggaaca atacattttt atccttacgt 1140

gacgtcttcg gcaaggattt aatctacaca ctgtattact ggaagtctag ctcctccggc 1200

aagaagaccg ccaagaccaa taccaacgaa tttttaattg acgtggacaa gggcgagaac 1260

tactgcttct ccgtgcaagc tgtgatcccc tcccggacag tgaaccggaa gtccaccgac 1320

tcccccgtgg agtgcatggg ccaagagaag ggagagtttc gtgagcagat cgtgctgacc 1380

cagtcccccg ctattatgag cgctagcccc ggtgaaaagg tgactatgac atgcagcgcc 1440

agctcttccg tgagctacat gaactggtat cagcagaagt ccggcaccag ccctaaaagg 1500

tggatctacg acaccagcaa gctggccagc ggcgtccccg ctcactttcg gggctccggc 1560

tccggaacaa gctactctct gaccatcagc ggcatggaag ccgaggatgc cgctacctat 1620

tactgtcagc agtggagctc caaccccttc acctttggat ccggcaccaa gctcgagatt 1680

aatcgtggag gcggaggtag cggaggaggc ggatccggcg gtggaggtag ccaagttcag 1740

ctccagcaaa gcggcgccga actcgctcgg cccggcgctt ccgtgaagat gtcttgtaag 1800

gcctccggct ataccttcac ccggtacaca atgcactggg tcaagcaacg gcccggtcaa 1860

ggtttagagt ggattggcta tatcaacccc tcccggggct ataccaacta caaccagaag 1920

ttcaaggaca aagccaccct caccaccgac aagtccagca gcaccgctta catgcagctg 1980

agctctttaa catccgagga ttccgccgtg tactactgcg ctcggtacta cgacgatcat 2040

tactgcctcg attactgggg ccaaggtacc accttaacag tctcctcc 2088

<210> 91

<211> 714

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 91

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly

20 25 30

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

35 40 45

Tyr Val Ile Gln Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp

50 55 60

Ile Gly Ser Ile Asn Pro Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys

65 70 75 80

Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala

85 90 95

Tyr Met Glu Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr

100 105 110

Cys Ala Arg Trp Gly Asp Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu

115 120 125

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Asp Ile Glu Met Thr Gln Ser Pro Ala Ile Met Ser Ala

145 150 155 160

Ser Leu Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val

165 170 175

Ser Ser Ser Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro

180 185 190

Lys Leu Cys Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro

195 200 205

Arg Phe Ser Gly Ser Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser

210 215 220

Met Glu Ala Glu Asp Ala Ala Thr Tyr Phe Cys His Gln Tyr His Arg

225 230 235 240

Ser Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg Ser Gly

245 250 255

Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn

260 265 270

Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr

275 280 285

Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe

290 295 300

Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp

305 310 315 320

Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn

325 330 335

Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro

340 345 350

Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln

355 360 365

Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu

370 375 380

Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val

385 390 395 400

Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser

405 410 415

Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp

420 425 430

Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro

435 440 445

Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met

450 455 460

Gly Gln Glu Lys Gly Glu Phe Arg Glu Gln Ile Val Leu Thr Gln Ser

465 470 475 480

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

485 490 495

Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

500 505 510

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser

515 520 525

Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser

530 535 540

Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

545 550 555 560

Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

565 570 575

Glu Ile Asn Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

580 585 590

Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

595 600 605

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

610 615 620

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

625 630 635 640

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

645 650 655

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

660 665 670

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

675 680 685

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp

690 695 700

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

705 710

<210> 92

<211> 2142

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 92

atgaaatggg tcaccttcat ctctttactg tttttattta gcagcgccta cagcgtgcag 60

ctgcagcagt ccggacccga actggtcaag cccggtgcct ccgtgaaaat gtcttgtaag 120

gcttctggct acacctttac ctcctacgtc atccaatggg tgaagcagaa gcccggtcaa 180

ggtctcgagt ggatcggcag catcaatccc tacaacgatt acaccaagta taacgaaaag 240

tttaagggca aggccactct gacaagcgac aagagctcca ttaccgccta catggagttt 300

tcctctttaa cttctgagga ctccgcttta tactattgcg ctcgttgggg cgatggcaat 360

tattggggcc ggggaactac tttaacagtg agctccggcg gcggcggaag cggaggtgga 420

ggatctggcg gtggaggcag cgacatcgag atgacacagt cccccgctat catgagcgcc 480

tctttaggag aacgtgtgac catgacttgt acagcttcct ccagcgtgag cagctcctat 540

ttccactggt accagcagaa acccggctcc tcccctaaac tgtgtatcta ctccacaagc 600

aatttagcta gcggcgtgcc tcctcgtttt agcggctccg gcagcacctc ttactcttta 660

accattagct ctatggaggc cgaagatgcc gccacatact tttgccatca gtaccaccgg 720

tcccctacct ttggcggagg cacaaagctg gagaccaagc ggagcggcac caccaacaca 780

gtggccgcct acaatctgac ttggaaatcc accaacttca agaccatcct cgagtgggag 840

cccaagcccg ttaatcaagt ttataccgtg cagatttcca ccaagagcgg cgactggaaa 900

tccaagtgct tctataccac agacaccgag tgcgatctca ccgacgagat cgtcaaagac 960

gtgaagcaga catatttagc tagggtgttc tcctaccccg ctggaaacgt ggagagcacc 1020

ggatccgctg gagagccttt atacgagaac tcccccgaat tcacccccta tctggaaacc 1080

aatttaggcc agcccaccat ccagagcttc gaacaagttg gcacaaaggt gaacgtcacc 1140

gtcgaagatg agaggacttt agtgcggagg aacaatacat ttttatcctt acgtgacgtc 1200

ttcggcaagg atttaatcta cacactgtat tactggaagt ctagctcctc cggcaagaag 1260

accgccaaga ccaataccaa cgaattttta attgacgtgg acaagggcga gaactactgc 1320

ttctccgtgc aagctgtgat cccctcccgg acagtgaacc ggaagtccac cgactccccc 1380

gtggagtgca tgggccaaga gaagggagag tttcgtgagc agatcgtgct gacccagtcc 1440

cccgctatta tgagcgctag ccccggtgaa aaggtgacta tgacatgcag cgccagctct 1500

tccgtgagct acatgaactg gtatcagcag aagtccggca ccagccctaa aaggtggatc 1560

tacgacacca gcaagctggc cagcggcgtc cccgctcact ttcggggctc cggctccgga 1620

acaagctact ctctgaccat cagcggcatg gaagccgagg atgccgctac ctattactgt 1680

cagcagtgga gctccaaccc cttcaccttt ggatccggca ccaagctcga gattaatcgt 1740

ggaggcggag gtagcggagg aggcggatcc ggcggtggag gtagccaagt tcagctccag 1800

caaagcggcg ccgaactcgc tcggcccggc gcttccgtga agatgtcttg taaggcctcc 1860

ggctatacct tcacccggta cacaatgcac tgggtcaagc aacggcccgg tcaaggttta 1920

gagtggattg gctatatcaa cccctcccgg ggctatacca actacaacca gaagttcaag 1980

gacaaagcca ccctcaccac cgacaagtcc agcagcaccg cttacatgca gctgagctct 2040

ttaacatccg aggattccgc cgtgtactac tgcgctcggt actacgacga tcattactgc 2100

ctcgattact ggggccaagg taccacctta acagtctcct cc 2142

<210> 93

<211> 219

<212> PRT

<213> Chile person

<400> 93

Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser

1 5 10 15

Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln

20 25 30

Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys

35 40 45

Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val

50 55 60

Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala

65 70 75 80

Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn

85 90 95

Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr

100 105 110

Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu

115 120 125

Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg

130 135 140

Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser

145 150 155 160

Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu

165 170 175

Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val

180 185 190

Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu

195 200 205

Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu

210 215

<210> 94

<211> 657

<212> DNA

<213> Chile person

<400> 94

agcggcacaa ccaacacagt cgctgcctat aacctcactt ggaagagcac caacttcaaa 60

accatcctcg aatgggaacc caaacccgtt aaccaagttt acaccgtgca gatcagcacc 120

aagtccggcg actggaagtc caaatgtttc tataccaccg acaccgagtg cgatctcacc 180

gatgagatcg tgaaagatgt gaaacagacc tacctcgccc gggtgtttag ctaccccgcc 240

ggcaatgtgg agagcactgg ttccgctggc gagcctttat acgagaacag ccccgaattt 300

accccttacc tcgagaccaa tttaggacag cccaccatcc aaagctttga gcaagttggc 360

acaaaggtga atgtgacagt ggaggacgag cggactttag tgcggcggaa caacaccttt 420

ctcagcctcc gggatgtgtt cggcaaagat ttaatctaca cactgtatta ctggaagtcc 480

tcttcctccg gcaagaagac agctaaaacc aacacaaacg agtttttaat cgacgtggat 540

aaaggcgaaa actactgttt cagcgtgcaa gctgtgatcc cctcccggac cgtgaatagg 600

aaaagcaccg atagccccgt tgagtgcatg ggccaagaaa agggcgagtt ccgggag 657

<210> 95

<211> 223

<212> PRT

<213> mouse (Mus sp.)

<400> 95

Ala Gly Ile Pro Glu Lys Ala Phe Asn Leu Thr Trp Ile Ser Thr Asp

1 5 10 15

Phe Lys Thr Ile Leu Glu Trp Gln Pro Lys Pro Thr Asn Tyr Thr Tyr

20 25 30

Thr Val Gln Ile Ser Asp Arg Ser Arg Asn Trp Lys Asn Lys Cys Phe

35 40 45

Ser Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp

50 55 60

Val Thr Trp Ala Tyr Glu Ala Lys Val Leu Ser Val Pro Arg Arg Asn

65 70 75 80

Ser Val His Gly Asp Gly Asp Gln Leu Val Ile His Gly Glu Glu Pro

85 90 95

Pro Phe Thr Asn Ala Pro Lys Phe Leu Pro Tyr Arg Asp Thr Asn Leu

100 105 110

Gly Gln Pro Val Ile Gln Gln Phe Glu Gln Asp Gly Arg Lys Leu Asn

115 120 125

Val Val Val Lys Asp Ser Leu Thr Leu Val Arg Lys Asn Gly Thr Phe

130 135 140

Leu Thr Leu Arg Gln Val Phe Gly Lys Asp Leu Gly Tyr Ile Ile Thr

145 150 155 160

Tyr Arg Lys Gly Ser Ser Thr Gly Lys Lys Thr Asn Ile Thr Asn Thr

165 170 175

Asn Glu Phe Ser Ile Asp Val Glu Glu Gly Val Ser Tyr Cys Phe Phe

180 185 190

Val Gln Ala Met Ile Phe Ser Arg Lys Thr Asn Gln Asn Ser Pro Gly

195 200 205

Ser Ser Thr Val Cys Thr Glu Gln Trp Lys Ser Phe Leu Gly Glu

210 215 220

<210> 96

<211> 224

<212> PRT

<213> rat (Rattus sp.)

<400> 96

Ala Gly Thr Pro Pro Gly Lys Ala Phe Asn Leu Thr Trp Ile Ser Thr

1 5 10 15

Asp Phe Lys Thr Ile Leu Glu Trp Gln Pro Lys Pro Thr Asn Tyr Thr

20 25 30

Tyr Thr Val Gln Ile Ser Asp Arg Ser Arg Asn Trp Lys Tyr Lys Cys

35 40 45

Thr Gly Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys

50 55 60

Asp Val Asn Trp Thr Tyr Glu Ala Arg Val Leu Ser Val Pro Trp Arg

65 70 75 80

Asn Ser Thr His Gly Lys Glu Thr Leu Phe Gly Thr His Gly Glu Glu

85 90 95

Pro Pro Phe Thr Asn Ala Arg Lys Phe Leu Pro Tyr Arg Asp Thr Lys

100 105 110

Ile Gly Gln Pro Val Ile Gln Lys Tyr Glu Gln Gly Gly Thr Lys Leu

115 120 125

Lys Val Thr Val Lys Asp Ser Phe Thr Leu Val Arg Lys Asn Gly Thr

130 135 140

Phe Leu Thr Leu Arg Gln Val Phe Gly Asn Asp Leu Gly Tyr Ile Leu

145 150 155 160

Thr Tyr Arg Lys Asp Ser Ser Thr Gly Arg Lys Thr Asn Thr Thr His

165 170 175

Thr Asn Glu Phe Leu Ile Asp Val Glu Lys Gly Val Ser Tyr Cys Phe

180 185 190

Phe Ala Gln Ala Val Ile Phe Ser Arg Lys Thr Asn His Lys Ser Pro

195 200 205

Glu Ser Ile Thr Lys Cys Thr Glu Gln Trp Lys Ser Val Leu Gly Glu

210 215 220

<210> 97

<211> 219

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 97

Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser

1 5 10 15

Thr Asn Phe Ala Thr Ala Leu Glu Trp Glu Pro Lys Pro Val Asn Gln

20 25 30

Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys

35 40 45

Cys Phe Tyr Thr Thr Asp Thr Glu Cys Ala Leu Thr Asp Glu Ile Val

50 55 60

Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala

65 70 75 80

Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn

85 90 95

Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr

100 105 110

Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu

115 120 125

Asp Glu Arg Thr Leu Val Ala Arg Asn Asn Thr Ala Leu Ser Leu Arg

130 135 140

Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser

145 150 155 160

Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu

165 170 175

Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val

180 185 190

Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu

195 200 205

Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu

210 215

<210> 98

<211> 219

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 98

Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser

1 5 10 15

Thr Asn Phe Ala Thr Ala Leu Glu Trp Glu Pro Lys Pro Val Asn Gln

20 25 30

Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Ala Lys Ser Lys

35 40 45

Cys Phe Tyr Thr Thr Asp Thr Glu Cys Ala Leu Thr Asp Glu Ile Val

50 55 60

Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala

65 70 75 80

Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Ala Glu Asn

85 90 95

Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr

100 105 110

Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu

115 120 125

Asp Glu Arg Thr Leu Val Ala Arg Asn Asn Thr Ala Leu Ser Leu Arg

130 135 140

Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser

145 150 155 160

Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu

165 170 175

Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val

180 185 190

Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu

195 200 205

Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu

210 215

<210> 99

<211> 4

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 99

Gly Gly Gly Ser

1

<210> 100

<211> 5

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 100

Gly Gly Gly Gly Ser

1 5

<210> 101

<211> 4

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 101

Gly Gly Ser Gly

1

<210> 102

<211> 15

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 102

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 103

<211> 45

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Oligonucleotide'

<400> 103

ggcggtggag gatccggagg aggtggctcc ggcggcggag gatct 45

<210> 104

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 104

Gly Gly Gly Ser Gly Gly Gly Ser

1 5

<210> 105

<211> 133

<212> PRT

<213> Chile person

<400> 105

Ala Pro Met Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp Val Asn Cys

1 5 10 15

Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu

20 25 30

Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu

35 40 45

Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala

50 55 60

Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn

65 70 75 80

Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro

85 90 95

Ile His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr

100 105 110

Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu

115 120 125

Ser Leu Ala Ile Phe

130

<210> 106

<211> 79

<212> PRT

<213> Chile person

<400> 106

Glu Gly Ala Val Leu Pro Arg Ser Ala Lys Glu Leu Arg Cys Gln Cys

1 5 10 15

Ile Lys Thr Tyr Ser Lys Pro Phe His Pro Lys Phe Ile Lys Glu Leu

20 25 30

Arg Val Ile Glu Ser Gly Pro His Cys Ala Asn Thr Glu Ile Ile Val

35 40 45

Lys Leu Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro Lys Glu Asn Trp

50 55 60

Val Gln Arg Val Val Glu Lys Phe Leu Lys Arg Ala Glu Asn Ser

65 70 75

<210> 107

<211> 160

<212> PRT

<213> Chile person

<400> 107

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

<210> 108

<211> 132

<212> PRT

<213> Chile person

<400> 108

Gly Ile Thr Ile Pro Arg Asn Pro Gly Cys Pro Asn Ser Glu Asp Lys

1 5 10 15

Asn Phe Pro Arg Thr Val Met Val Asn Leu Asn Ile His Asn Arg Asn

20 25 30

Thr Asn Thr Asn Pro Lys Arg Ser Ser Asp Tyr Tyr Asn Arg Ser Thr

35 40 45

Ser Pro Trp Asn Leu His Arg Asn Glu Asp Pro Glu Arg Tyr Pro Ser

50 55 60

Val Ile Trp Glu Ala Lys Cys Arg His Leu Gly Cys Ile Asn Ala Asp

65 70 75 80

Gly Asn Val Asp Tyr His Met Asn Ser Val Pro Ile Gln Gln Glu Ile

85 90 95

Leu Val Leu Arg Arg Glu Pro Pro His Cys Pro Asn Ser Phe Arg Leu

100 105 110

Glu Lys Ile Leu Val Ser Val Gly Cys Thr Cys Val Thr Pro Ile Val

115 120 125

His His Val Ala

130

<210> 109

<211> 157

<212> PRT

<213> Chile person

<400> 109

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 110

<211> 352

<212> PRT

<213> Chile person

<400> 110

Arg Asp Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Lys Ala Leu Arg

1 5 10 15

Asn Ala Asn Leu Arg Arg Asp Glu Ser Asn His Leu Thr Asp Leu Tyr

20 25 30

Arg Arg Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Tyr Val Gln Ser

35 40 45

Pro Arg Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Leu Thr Trp Arg

50 55 60

Leu His Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Phe Asp Asn Gln

65 70 75 80

Phe Gly Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Tyr Asp Phe Val

85 90 95

Glu Val Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Arg Gly Arg Trp

100 105 110

Cys Gly His Lys Glu Val Pro Pro Arg Ile Lys Ser Arg Thr Asn Gln

115 120 125

Ile Lys Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Ala Lys Pro Gly

130 135 140

Phe Lys Ile Tyr Tyr Ser Leu Leu Glu Asp Phe Gln Pro Ala Ala Ala

145 150 155 160

Ser Glu Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Ser Gly Val Ser

165 170 175

Tyr Asn Ser Pro Ser Val Thr Asp Pro Thr Leu Ile Ala Asp Ala Leu

180 185 190

Asp Lys Lys Ile Ala Glu Phe Asp Thr Val Glu Asp Leu Leu Lys Tyr

195 200 205

Phe Asn Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Met Tyr Leu Asp

210 215 220

Thr Pro Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Lys Ser Lys Val

225 230 235 240

Asp Leu Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Ser Cys Thr Pro

245 250 255

Arg Asn Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys Leu Ala Asn Val

260 265 270

Val Phe Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gly Gly Asn Cys

275 280 285

Gly Cys Gly Thr Val Asn Trp Arg Ser Cys Thr Cys Asn Ser Gly Lys

290 295 300

Thr Val Lys Lys Tyr His Glu Val Leu Gln Phe Glu Pro Gly His Ile

305 310 315 320

Lys Arg Arg Gly Arg Ala Lys Thr Met Ala Leu Val Asp Ile Gln Leu

325 330 335

Asp His His Glu Arg Cys Asp Cys Ile Cys Ser Ser Arg Pro Pro Arg

340 345 350

<210> 111

<211> 248

<212> PRT

<213> Chile person

<400> 111

Glu Gly Ile Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr

1 5 10 15

Lys Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr

20 25 30

Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met

35 40 45

Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe Ser

50 55 60

Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu Val

65 70 75 80

Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser Lys

85 90 95

Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe Thr Pro

100 105 110

Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp Ala Phe Lys Asp

115 120 125

Phe Val Val Ala Ser Glu Thr Ser Asp Cys Val Val Ser Ser Thr Leu

130 135 140

Ser Pro Glu Lys Asp Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu

145 150 155 160

Pro Pro Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn

165 170 175

Arg Lys Ala Lys Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala

180 185 190

Met Ala Leu Pro Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly

195 200 205

Ala Leu Tyr Trp Lys Lys Arg Gln Pro Ser Leu Thr Arg Ala Val Glu

210 215 220

Asn Ile Gln Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu

225 230 235 240

Lys Glu Arg Glu Phe Gln Glu Val

245

<210> 112

<211> 209

<212> PRT

<213> Chile person

<400> 112

Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala

1 5 10 15

Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val

20 25 30

Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp

35 40 45

Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala

50 55 60

Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His

65 70 75 80

Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe

85 90 95

Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu

100 105 110

Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu

115 120 125

Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser

130 135 140

Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro Pro Leu

145 150 155 160

Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu Leu Leu Ala Ala Ala

165 170 175

Trp Cys Leu His Trp Gln Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly

180 185 190

Glu Gln Val Pro Pro Val Pro Ser Pro Gln Asp Leu Leu Leu Val Glu

195 200 205

His

<210> 113

<211> 65

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 113

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg

65

<210> 114

<211> 195

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 114

attacatgcc cccctcccat gagcgtggag cacgccgaca tctgggtgaa gagctatagc 60

ctctacagcc gggagaggta tatctgtaac agcggcttca agaggaaggc cggcaccagc 120

agcctcaccg agtgcgtgct gaataaggct accaacgtgg ctcactggac aacaccctct 180

ttaaagtgca tccgg 195

<210> 115

<211> 114

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 115

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 116

<211> 342

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 116

aactgggtga acgtcatcag cgatttaaag aagatcgaag atttaattca gtccatgcat 60

atcgacgcca ctttatacac agaatccgac gtgcacccct cttgtaaggt gaccgccatg 120

aaatgttttt tactggagct gcaagttatc tctttagaga gcggagacgc tagcatccac 180

gacaccgtgg agaatttaat cattttagcc aataactctt tatccagcaa cggcaacgtg 240

acagagtccg gctgcaagga gtgcgaagag ctggaggaga agaacatcaa ggagtttctg 300

caatcctttg tgcacattgt ccagatgttc atcaatacct cc 342

<210> 117

<211> 18

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 117

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser

<210> 118

<211> 54

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Oligonucleotide'

<400> 118

atgaaatggg tgacctttat ttctttactg ttcctcttta gcagcgccta ctcc 54

<210> 119

<211> 54

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Oligonucleotide'

<400> 119

atgaagtggg tcacatttat ctctttactg ttcctcttct ccagcgccta cagc 54

<210> 120

<211> 54

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Oligonucleotide'

<400> 120

atgaaatggg tgacctttat ttctttactg ttcctcttta gcagcgccta ctcc 54

<210> 121

<211> 16

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 121

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Leu Gly Val Asn Cys

1 5 10 15

<210> 122

<211> 58

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 122

Met Gly Gln Ile Val Thr Met Phe Glu Ala Leu Pro His Ile Ile Asp

1 5 10 15

Glu Val Ile Asn Ile Val Ile Ile Val Leu Ile Ile Ile Thr Ser Ile

20 25 30

Lys Ala Val Tyr Asn Phe Ala Thr Cys Gly Ile Leu Ala Leu Val Ser

35 40 45

Phe Leu Phe Leu Ala Gly Arg Ser Cys Gly

50 55

<210> 123

<211> 97

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 123

Met Pro Asn His Gln Ser Gly Ser Pro Thr Gly Ser Ser Asp Leu Leu

1 5 10 15

Leu Ser Gly Lys Lys Gln Arg Pro His Leu Ala Leu Arg Arg Lys Arg

20 25 30

Arg Arg Glu Met Arg Lys Ile Asn Arg Lys Val Arg Arg Met Asn Leu

35 40 45

Ala Pro Ile Lys Glu Lys Thr Ala Trp Gln His Leu Gln Ala Leu Ile

50 55 60

Ser Glu Ala Glu Glu Val Leu Lys Thr Ser Gln Thr Pro Gln Asn Ser

65 70 75 80

Leu Thr Leu Phe Leu Ala Leu Leu Ser Val Leu Gly Pro Pro Val Thr

85 90 95

Gly

<210> 124

<211> 30

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 124

Met Asp Ser Lys Gly Ser Ser Gln Lys Gly Ser Arg Leu Leu Leu Leu

1 5 10 15

Leu Val Val Ser Asn Leu Leu Leu Cys Gln Gly Val Val Ser

20 25 30

<210> 125

<211> 16

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 125

Met Lys Cys Leu Leu Tyr Leu Ala Phe Leu Phe Leu Gly Val Asn Cys

1 5 10 15

<210> 126

<211> 15

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 126

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

1 5 10 15

<210> 127

<211> 26

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 127

Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg

1 5 10 15

Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu

20 25

<210> 128

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 128

Glu Glu Glu Glu Glu Glu

1 5

<210> 129

<211> 13

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 129

Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg

1 5 10

<210> 130

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 130

Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

<210> 131

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 131

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

<210> 132

<211> 5

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 132

His His His His His

1 5

<210> 133

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 133

His His His His His His

1 5

<210> 134

<211> 7

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 134

His His His His His His His

1 5

<210> 135

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 135

His His His His His His His His

1 5

<210> 136

<211> 9

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 136

His His His His His His His His His

1 5

<210> 137

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 137

His His His His His His His His His His

1 5 10

<210> 138

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 138

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 139

<211> 18

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 139

Thr Lys Glu Asn Pro Arg Ser Asn Gln Glu Glu Ser Tyr Asp Asp Asn

1 5 10 15

Glu Ser

<210> 140

<211> 15

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 140

Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser

1 5 10 15

<210> 141

<211> 38

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 141

Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly

1 5 10 15

Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro

20 25 30

Gln Gly Gln Arg Glu Pro

35

<210> 142

<211> 13

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 142

Ser Leu Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser

1 5 10

<210> 143

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 143

Thr Gln Asp Pro Ser Arg Val Gly

1 5

<210> 144

<211> 12

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 144

Pro Asp Arg Val Arg Ala Val Ser His Trp Ser Ser

1 5 10

<210> 145

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 145

Trp Ser His Pro Gln Phe Glu Lys

1 5

<210> 146

<211> 6

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 146

Cys Cys Pro Gly Cys Cys

1 5

<210> 147

<211> 10

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 147

Glu Val His Thr Asn Gln Asp Pro Leu Asp

1 5 10

<210> 148

<211> 14

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 148

Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr

1 5 10

<210> 149

<211> 11

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 149

Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys

1 5 10

<210> 150

<211> 8

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 150

Asp Leu Tyr Asp Asp Asp Asp Lys

1 5

<210> 151

<211> 241

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 151

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

145 150 155 160

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

165 170 175

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

180 185 190

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

210 215 220

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

225 230 235 240

Ser

<210> 152

<211> 723

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 152

cagatcgtgc tgacccaaag ccccgccatc atgagcgcta gccccggtga gaaggtgacc 60

atgacatgct ccgcttccag ctccgtgtcc tacatgaact ggtatcagca gaaaagcgga 120

accagcccca aaaggtggat ctacgacacc agcaagctgg cctccggagt gcccgctcat 180

ttccggggct ctggatccgg caccagctac tctttaacca tttccggcat ggaagctgaa 240

gacgctgcca cctactattg ccagcaatgg agcagcaacc ccttcacatt cggatctggc 300

accaagctcg aaatcaatcg tggaggaggt ggcagcggcg gcggtggatc cggcggagga 360

ggaagccaag ttcaactcca gcagagcggc gctgaactgg cccggcccgg cgcctccgtc 420

aagatgagct gcaaggcttc cggctataca tttactcgtt acacaatgca ttgggtcaag 480

cagaggcccg gtcaaggttt agagtggatc ggatatatca acccttcccg gggctacacc 540

aactataacc aaaagttcaa ggataaagcc actttaacca ctgacaagag ctcctccacc 600

gcctacatgc agctgtcctc tttaaccagc gaggactccg ctgtttacta ctgcgctagg 660

tattacgacg accactactg tttagactat tggggacaag gtaccacttt aaccgtcagc 720

agc 723

<210> 153

<211> 236

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 153

Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser

1 5 10 15

Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val

20 25 30

Ile Gln Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly

35 40 45

Ser Ile Asn Pro Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys Phe Lys

50 55 60

Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala Tyr Met

65 70 75 80

Glu Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys Ala

85 90 95

Arg Trp Gly Asp Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val

100 105 110

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Asp Ile Glu Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu

130 135 140

Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser

145 150 155 160

Ser Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu

165 170 175

Cys Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe

180 185 190

Ser Gly Ser Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

195 200 205

Ala Glu Asp Ala Ala Thr Tyr Phe Cys His Gln Tyr His Arg Ser Pro

210 215 220

Thr Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg

225 230 235

<210> 154

<211> 708

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 154

gtccagctgc agcagagcgg acccgaactc gtgaaacccg gtgcttccgt gaaaatgtct 60

tgtaaggcca gcggatacac cttcacctcc tatgtgatcc agtgggtcaa acagaagccc 120

ggacaaggtc tcgagtggat cggcagcatc aacccttaca acgactatac caaatacaac 180

gagaagttta agggaaaggc tactttaacc tccgacaaaa gctccatcac agcctacatg 240

gagttcagct ctttaacatc cgaggacagc gctctgtact attgcgcccg gtggggcgac 300

ggcaattact ggggacgggg cacaacactg accgtgagca gcggaggcgg aggctccggc 360

ggaggcggat ctggcggtgg cggctccgac atcgagatga cccagtcccc cgctatcatg 420

tccgcctctt taggcgagcg ggtcacaatg acttgtacag cctcctccag cgtctcctcc 480

tcctacttcc attggtacca acagaaaccc ggaagctccc ctaaactgtg catctacagc 540

accagcaatc tcgccagcgg cgtgccccct aggttttccg gaagcggaag caccagctac 600

tctttaacca tctcctccat ggaggctgag gatgccgcca cctacttttg tcaccagtac 660

caccggtccc ccaccttcgg aggcggcacc aaactggaga caaagagg 708

<210> 155

<211> 696

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 155

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln

115 120 125

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

130 135 140

Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

145 150 155 160

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

165 170 175

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

180 185 190

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

195 200 205

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

210 215 220

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

225 230 235 240

Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys

245 250 255

Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn

260 265 270

Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser

275 280 285

Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile

290 295 300

Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro

305 310 315 320

Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu

325 330 335

Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro

340 345 350

Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val

355 360 365

Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu

370 375 380

Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys

385 390 395 400

Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe

405 410 415

Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala

420 425 430

Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val

435 440 445

Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Val Gln Leu Gln

450 455 460

Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser

465 470 475 480

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Ile Gln Trp Val

485 490 495

Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Ser Ile Asn Pro

500 505 510

Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr

515 520 525

Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala Tyr Met Glu Phe Ser Ser

530 535 540

Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys Ala Arg Trp Gly Asp

545 550 555 560

Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

565 570 575

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu

580 585 590

Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu Arg Val

595 600 605

Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Phe His

610 615 620

Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Cys Ile Tyr Ser

625 630 635 640

Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser Gly Ser Gly

645 650 655

Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala

660 665 670

Ala Thr Tyr Phe Cys His Gln Tyr His Arg Ser Pro Thr Phe Gly Gly

675 680 685

Gly Thr Lys Leu Glu Thr Lys Arg

690 695

<210> 156

<211> 2088

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 156

cagatcgtgc tgacccaaag ccccgccatc atgagcgcta gccccggtga gaaggtgacc 60

atgacatgct ccgcttccag ctccgtgtcc tacatgaact ggtatcagca gaaaagcgga 120

accagcccca aaaggtggat ctacgacacc agcaagctgg cctccggagt gcccgctcat 180

ttccggggct ctggatccgg caccagctac tctttaacca tttccggcat ggaagctgaa 240

gacgctgcca cctactattg ccagcaatgg agcagcaacc ccttcacatt cggatctggc 300

accaagctcg aaatcaatcg tggaggaggt ggcagcggcg gcggtggatc cggcggagga 360

ggaagccaag ttcaactcca gcagagcggc gctgaactgg cccggcccgg cgcctccgtc 420

aagatgagct gcaaggcttc cggctataca tttactcgtt acacaatgca ttgggtcaag 480

cagaggcccg gtcaaggttt agagtggatc ggatatatca acccttcccg gggctacacc 540

aactataacc aaaagttcaa ggataaagcc actttaacca ctgacaagag ctcctccacc 600

gcctacatgc agctgtcctc tttaaccagc gaggactccg ctgtttacta ctgcgctagg 660

tattacgacg accactactg tttagactat tggggacaag gtaccacttt aaccgtcagc 720

agctccggca ccaccaatac cgtggccgct tataacctca catggaagag caccaacttc 780

aagacaattc tggaatggga acccaagccc gtcaatcaag tttacaccgt gcagatctcc 840

accaaatccg gagactggaa gagcaagtgc ttctacacaa cagacaccga gtgtgattta 900

accgacgaaa tcgtcaagga cgtcaagcaa acctatctgg ctcgggtctt ttcctacccc 960

gctggcaatg tcgagtccac cggctccgct ggcgagcctc tctacgagaa ttcccccgaa 1020

ttcacccctt atttagagac caatttaggc cagcctacca tccagagctt cgagcaagtt 1080

ggcaccaagg tgaacgtcac cgtcgaggat gaaaggactt tagtgcggcg gaataacaca 1140

tttttatccc tccgggatgt gttcggcaaa gacctcatct acacactgta ctattggaag 1200

tccagctcct ccggcaaaaa gaccgctaag accaacacca acgagttttt aattgacgtg 1260

gacaaaggcg agaactactg cttcagcgtg caagccgtga tcccttctcg taccgtcaac 1320

cggaagagca cagattcccc cgttgagtgc atgggccaag aaaagggcga gttccgggag 1380

gtccagctgc agcagagcgg acccgaactc gtgaaacccg gtgcttccgt gaaaatgtct 1440

tgtaaggcca gcggatacac cttcacctcc tatgtgatcc agtgggtcaa acagaagccc 1500

ggacaaggtc tcgagtggat cggcagcatc aacccttaca acgactatac caaatacaac 1560

gagaagttta agggaaaggc tactttaacc tccgacaaaa gctccatcac agcctacatg 1620

gagttcagct ctttaacatc cgaggacagc gctctgtact attgcgcccg gtggggcgac 1680

ggcaattact ggggacgggg cacaacactg accgtgagca gcggaggcgg aggctccggc 1740

ggaggcggat ctggcggtgg cggctccgac atcgagatga cccagtcccc cgctatcatg 1800

tccgcctctt taggcgagcg ggtcacaatg acttgtacag cctcctccag cgtctcctcc 1860

tcctacttcc attggtacca acagaaaccc ggaagctccc ctaaactgtg catctacagc 1920

accagcaatc tcgccagcgg cgtgccccct aggttttccg gaagcggaag caccagctac 1980

tctttaacca tctcctccat ggaggctgag gatgccgcca cctacttttg tcaccagtac 2040

caccggtccc ccaccttcgg aggcggcacc aaactggaga caaagagg 2088

<210> 157

<211> 714

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 157

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser

20 25 30

Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser

35 40 45

Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp

50 55 60

Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg

65 70 75 80

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu

85 90 95

Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro

100 105 110

Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

130 135 140

Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met

145 150 155 160

Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

165 170 175

Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn

180 185 190

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala

195 200 205

Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser

210 215 220

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

225 230 235 240

Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

245 250 255

Val Ser Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr

260 265 270

Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro

275 280 285

Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp

290 295 300

Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp

305 310 315 320

Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser

325 330 335

Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu

340 345 350

Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly

355 360 365

Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val

370 375 380

Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu

385 390 395 400

Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr

405 410 415

Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn

420 425 430

Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val

435 440 445

Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser

450 455 460

Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Val Gln

465 470 475 480

Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys

485 490 495

Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Ile Gln

500 505 510

Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Ser Ile

515 520 525

Asn Pro Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys

530 535 540

Ala Thr Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala Tyr Met Glu Phe

545 550 555 560

Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr Cys Ala Arg Trp

565 570 575

Gly Asp Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu Thr Val Ser Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

595 600 605

Ile Glu Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly Glu

610 615 620

Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr

625 630 635 640

Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Cys Ile

645 650 655

Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro Arg Phe Ser Gly

660 665 670

Ser Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

675 680 685

Asp Ala Ala Thr Tyr Phe Cys His Gln Tyr His Arg Ser Pro Thr Phe

690 695 700

Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg

705 710

<210> 158

<211> 2142

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 158

atgaagtggg tgaccttcat cagcttatta tttttattca gctccgccta ttcccagatc 60

gtgctgaccc aaagccccgc catcatgagc gctagccccg gtgagaaggt gaccatgaca 120

tgctccgctt ccagctccgt gtcctacatg aactggtatc agcagaaaag cggaaccagc 180

cccaaaaggt ggatctacga caccagcaag ctggcctccg gagtgcccgc tcatttccgg 240

ggctctggat ccggcaccag ctactcttta accatttccg gcatggaagc tgaagacgct 300

gccacctact attgccagca atggagcagc aaccccttca cattcggatc tggcaccaag 360

ctcgaaatca atcgtggagg aggtggcagc ggcggcggtg gatccggcgg aggaggaagc 420

caagttcaac tccagcagag cggcgctgaa ctggcccggc ccggcgcctc cgtcaagatg 480

agctgcaagg cttccggcta tacatttact cgttacacaa tgcattgggt caagcagagg 540

cccggtcaag gtttagagtg gatcggatat atcaaccctt cccggggcta caccaactat 600

aaccaaaagt tcaaggataa agccacttta accactgaca agagctcctc caccgcctac 660

atgcagctgt cctctttaac cagcgaggac tccgctgttt actactgcgc taggtattac 720

gacgaccact actgtttaga ctattgggga caaggtacca ctttaaccgt cagcagctcc 780

ggcaccacca ataccgtggc cgcttataac ctcacatgga agagcaccaa cttcaagaca 840

attctggaat gggaacccaa gcccgtcaat caagtttaca ccgtgcagat ctccaccaaa 900

tccggagact ggaagagcaa gtgcttctac acaacagaca ccgagtgtga tttaaccgac 960

gaaatcgtca aggacgtcaa gcaaacctat ctggctcggg tcttttccta ccccgctggc 1020

aatgtcgagt ccaccggctc cgctggcgag cctctctacg agaattcccc cgaattcacc 1080

ccttatttag agaccaattt aggccagcct accatccaga gcttcgagca agttggcacc 1140

aaggtgaacg tcaccgtcga ggatgaaagg actttagtgc ggcggaataa cacattttta 1200

tccctccggg atgtgttcgg caaagacctc atctacacac tgtactattg gaagtccagc 1260

tcctccggca aaaagaccgc taagaccaac accaacgagt ttttaattga cgtggacaaa 1320

ggcgagaact actgcttcag cgtgcaagcc gtgatccctt ctcgtaccgt caaccggaag 1380

agcacagatt cccccgttga gtgcatgggc caagaaaagg gcgagttccg ggaggtccag 1440

ctgcagcaga gcggacccga actcgtgaaa cccggtgctt ccgtgaaaat gtcttgtaag 1500

gccagcggat acaccttcac ctcctatgtg atccagtggg tcaaacagaa gcccggacaa 1560

ggtctcgagt ggatcggcag catcaaccct tacaacgact ataccaaata caacgagaag 1620

tttaagggaa aggctacttt aacctccgac aaaagctcca tcacagccta catggagttc 1680

agctctttaa catccgagga cagcgctctg tactattgcg cccggtgggg cgacggcaat 1740

tactggggac ggggcacaac actgaccgtg agcagcggag gcggaggctc cggcggaggc 1800

ggatctggcg gtggcggctc cgacatcgag atgacccagt cccccgctat catgtccgcc 1860

tctttaggcg agcgggtcac aatgacttgt acagcctcct ccagcgtctc ctcctcctac 1920

ttccattggt accaacagaa acccggaagc tcccctaaac tgtgcatcta cagcaccagc 1980

aatctcgcca gcggcgtgcc ccctaggttt tccggaagcg gaagcaccag ctactcttta 2040

accatctcct ccatggaggc tgaggatgcc gccacctact tttgtcacca gtaccaccgg 2100

tcccccacct tcggaggcgg caccaaactg gagacaaaga gg 2142

<210> 159

<211> 399

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 159

gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60

ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120

acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180

gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240

agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300

acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360

tggattacct tttgtcaaag catcatctca acactaact 399

<210> 160

<211> 399

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 160

gcccccacct cctcctccac caagaagacc cagctgcagc tggagcattt actgctggat 60

ttacagatga ttttaaacgg catcaacaac tacaagaacc ccaagctgac tcgtatgctg 120

accttcaagt tctacatgcc caagaaggcc accgagctga agcatttaca gtgtttagag 180

gaggagctga agcccctcga ggaggtgctg aatttagccc agtccaagaa tttccattta 240

aggccccggg atttaatcag caacatcaac gtgatcgttt tagagctgaa gggctccgag 300

accaccttca tgtgcgagta cgccgacgag accgccacca tcgtggagtt tttaaatcgt 360

tggatcacct tctgccagtc catcatctcc actttaacc 399

<210> 161

<211> 485

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 161

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn

130 135 140

Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro

145 150 155 160

Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly

165 170 175

Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu

180 185 190

Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val

195 200 205

Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu

210 215 220

Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn

225 230 235 240

Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val

245 250 255

Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr

260 265 270

Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu

275 280 285

Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn

290 295 300

Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe

305 310 315 320

Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr

325 330 335

Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu

340 345 350

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

355 360 365

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

370 375 380

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

385 390 395 400

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

405 410 415

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

420 425 430

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

435 440 445

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

450 455 460

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

465 470 475 480

Ile Ser Thr Leu Thr

485

<210> 162

<211> 1455

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 162

gcccccacct cctcctccac caagaagacc cagctgcagc tggagcattt actgctggat 60

ttacagatga ttttaaacgg catcaacaac tacaagaacc ccaagctgac tcgtatgctg 120

accttcaagt tctacatgcc caagaaggcc accgagctga agcatttaca gtgtttagag 180

gaggagctga agcccctcga ggaggtgctg aatttagccc agtccaagaa tttccattta 240

aggccccggg atttaatcag caacatcaac gtgatcgttt tagagctgaa gggctccgag 300

accaccttca tgtgcgagta cgccgacgag accgccacca tcgtggagtt tttaaatcgt 360

tggatcacct tctgccagtc catcatctcc actttaacca gcggcacaac caacacagtc 420

gctgcctata acctcacttg gaagagcacc aacttcaaaa ccatcctcga atgggaaccc 480

aaacccgtta accaagttta caccgtgcag atcagcacca agtccggcga ctggaagtcc 540

aaatgtttct ataccaccga caccgagtgc gatctcaccg atgagatcgt gaaagatgtg 600

aaacagacct acctcgcccg ggtgtttagc taccccgccg gcaatgtgga gagcactggt 660

tccgctggcg agcctttata cgagaacagc cccgaattta ccccttacct cgagaccaat 720

ttaggacagc ccaccatcca aagctttgag caagttggca caaaggtgaa tgtgacagtg 780

gaggacgagc ggactttagt gcggcggaac aacacctttc tcagcctccg ggatgtgttc 840

ggcaaagatt taatctacac actgtattac tggaagtcct cttcctccgg caagaagaca 900

gctaaaacca acacaaacga gtttttaatc gacgtggata aaggcgaaaa ctactgtttc 960

agcgtgcaag ctgtgatccc ctcccggacc gtgaatagga aaagcaccga tagccccgtt 1020

gagtgcatgg gccaagaaaa gggcgagttc cgggaggcac ctacttcaag ttctacaaag 1080

aaaacacagc tacaactgga gcatttactg ctggatttac agatgatttt gaatggaatt 1140

aataattaca agaatcccaa actcaccagg atgctcacat ttaagtttta catgcccaag 1200

aaggccacag aactgaaaca tcttcagtgt ctagaagaag aactcaaacc tctggaggaa 1260

gtgctaaatt tagctcaaag caaaaacttt cacttaagac ccagggactt aatcagcaat 1320

atcaacgtaa tagttctgga actaaaggga tctgaaacaa cattcatgtg tgaatatgct 1380

gatgagacag caaccattgt agaatttctg aacagatgga ttaccttttg tcaaagcatc 1440

atctcaacac taact 1455

<210> 163

<211> 503

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 163

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu

20 25 30

Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn

35 40 45

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

50 55 60

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

65 70 75 80

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe

85 90 95

His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu

100 105 110

Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu

115 120 125

Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln

130 135 140

Ser Ile Ile Ser Thr Leu Thr Ser Gly Thr Thr Asn Thr Val Ala Ala

145 150 155 160

Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp

165 170 175

Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys

180 185 190

Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys

195 200 205

Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala

210 215 220

Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala

225 230 235 240

Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu

245 250 255

Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr

260 265 270

Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn

275 280 285

Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr

290 295 300

Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys

305 310 315 320

Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr

325 330 335

Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys

340 345 350

Ser Thr Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe

355 360 365

Arg Glu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu

370 375 380

Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn

385 390 395 400

Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met

405 410 415

Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu

420 425 430

Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe

435 440 445

His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu

450 455 460

Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu

465 470 475 480

Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln

485 490 495

Ser Ile Ile Ser Thr Leu Thr

500

<210> 164

<211> 1509

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 164

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccgccccc 60

acctcctcct ccaccaagaa gacccagctg cagctggagc atttactgct ggatttacag 120

atgattttaa acggcatcaa caactacaag aaccccaagc tgactcgtat gctgaccttc 180

aagttctaca tgcccaagaa ggccaccgag ctgaagcatt tacagtgttt agaggaggag 240

ctgaagcccc tcgaggaggt gctgaattta gcccagtcca agaatttcca tttaaggccc 300

cgggatttaa tcagcaacat caacgtgatc gttttagagc tgaagggctc cgagaccacc 360

ttcatgtgcg agtacgccga cgagaccgcc accatcgtgg agtttttaaa tcgttggatc 420

accttctgcc agtccatcat ctccacttta accagcggca caaccaacac agtcgctgcc 480

tataacctca cttggaagag caccaacttc aaaaccatcc tcgaatggga acccaaaccc 540

gttaaccaag tttacaccgt gcagatcagc accaagtccg gcgactggaa gtccaaatgt 600

ttctatacca ccgacaccga gtgcgatctc accgatgaga tcgtgaaaga tgtgaaacag 660

acctacctcg cccgggtgtt tagctacccc gccggcaatg tggagagcac tggttccgct 720

ggcgagcctt tatacgagaa cagccccgaa tttacccctt acctcgagac caatttagga 780

cagcccacca tccaaagctt tgagcaagtt ggcacaaagg tgaatgtgac agtggaggac 840

gagcggactt tagtgcggcg gaacaacacc tttctcagcc tccgggatgt gttcggcaaa 900

gatttaatct acacactgta ttactggaag tcctcttcct ccggcaagaa gacagctaaa 960

accaacacaa acgagttttt aatcgacgtg gataaaggcg aaaactactg tttcagcgtg 1020

caagctgtga tcccctcccg gaccgtgaat aggaaaagca ccgatagccc cgttgagtgc 1080

atgggccaag aaaagggcga gttccgggag gcacctactt caagttctac aaagaaaaca 1140

cagctacaac tggagcattt actgctggat ttacagatga ttttgaatgg aattaataat 1200

tacaagaatc ccaaactcac caggatgctc acatttaagt tttacatgcc caagaaggcc 1260

acagaactga aacatcttca gtgtctagaa gaagaactca aacctctgga ggaagtgcta 1320

aatttagctc aaagcaaaaa ctttcactta agacccaggg acttaatcag caatatcaac 1380

gtaatagttc tggaactaaa gggatctgaa acaacattca tgtgtgaata tgctgatgag 1440

acagcaacca ttgtagaatt tctgaacaga tggattacct tttgtcaaag catcatctca 1500

acactaact 1509

<210> 165

<211> 342

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 165

aactgggtga acgtgatcag cgatttaaag aagatcgagg atttaatcca gagcatgcac 60

atcgacgcca ctctgtacac tgagagcgac gtgcacccta gctgcaaggt gactgccatg 120

aagtgctttt tactggagct gcaagttatc tctttagaga gcggcgatgc cagcatccac 180

gacactgtgg agaatttaat cattttagcc aacaactctt taagcagcaa cggcaacgtg 240

acagagagcg gctgcaagga gtgcgaggag ctggaggaga agaacatcaa ggagttttta 300

cagagcttcg tgcacatcgt gcagatgttc atcaacacta gc 342

<210> 166

<211> 342

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 166

aactgggtga acgtcatcag cgatttaaag aagatcgaag atttaattca gtccatgcat 60

atcgacgcca ctttatacac agaatccgac gtgcacccct cttgtaaggt gaccgccatg 120

aaatgttttt tactggagct gcaagttatc tctttagaga gcggagacgc tagcatccac 180

gacaccgtgg agaatttaat cattttagcc aataactctt tatccagcaa cggcaacgtg 240

acagagtccg gctgcaagga gtgcgaagag ctggaggaga agaacatcaa ggagtttctg 300

caatcctttg tgcacattgt ccagatgttc atcaatacct cc 342

<210> 167

<211> 447

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 167

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp

115 120 125

Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val

130 135 140

Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys

145 150 155 160

Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu

165 170 175

Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr

180 185 190

Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr

195 200 205

Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln

210 215 220

Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr

225 230 235 240

Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser

245 250 255

Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp

260 265 270

Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu

275 280 285

Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln

290 295 300

Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro

305 310 315 320

Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Asn Trp Val

325 330 335

Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met

340 345 350

His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys

355 360 365

Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser

370 375 380

Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile

385 390 395 400

Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser

405 410 415

Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe

420 425 430

Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

435 440 445

<210> 168

<211> 1341

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 168

aactgggtga acgtgatcag cgatttaaag aagatcgagg atttaatcca gagcatgcac 60

atcgacgcca ctctgtacac tgagagcgac gtgcacccta gctgcaaggt gactgccatg 120

aagtgctttt tactggagct gcaagttatc tctttagaga gcggcgatgc cagcatccac 180

gacactgtgg agaatttaat cattttagcc aacaactctt taagcagcaa cggcaacgtg 240

acagagagcg gctgcaagga gtgcgaggag ctggaggaga agaacatcaa ggagttttta 300

cagagcttcg tgcacatcgt gcagatgttc atcaacacta gcagcggcac aaccaacaca 360

gtcgctgcct ataacctcac ttggaagagc accaacttca aaaccatcct cgaatgggaa 420

cccaaacccg ttaaccaagt ttacaccgtg cagatcagca ccaagtccgg cgactggaag 480

tccaaatgtt tctataccac cgacaccgag tgcgatctca ccgatgagat cgtgaaagat 540

gtgaaacaga cctacctcgc ccgggtgttt agctaccccg ccggcaatgt ggagagcact 600

ggttccgctg gcgagccttt atacgagaac agccccgaat ttacccctta cctcgagacc 660

aatttaggac agcccaccat ccaaagcttt gagcaagttg gcacaaaggt gaatgtgaca 720

gtggaggacg agcggacttt agtgcggcgg aacaacacct ttctcagcct ccgggatgtg 780

ttcggcaaag atttaatcta cacactgtat tactggaagt cctcttcctc cggcaagaag 840

acagctaaaa ccaacacaaa cgagttttta atcgacgtgg ataaaggcga aaactactgt 900

ttcagcgtgc aagctgtgat cccctcccgg accgtgaata ggaaaagcac cgatagcccc 960

gttgagtgca tgggccaaga aaagggcgag ttccgggaga actgggtgaa cgtcatcagc 1020

gatttaaaga agatcgaaga tttaattcag tccatgcata tcgacgccac tttatacaca 1080

gaatccgacg tgcacccctc ttgtaaggtg accgccatga aatgtttttt actggagctg 1140

caagttatct ctttagagag cggagacgct agcatccacg acaccgtgga gaatttaatc 1200

attttagcca ataactcttt atccagcaac ggcaacgtga cagagtccgg ctgcaaggag 1260

tgcgaagagc tggaggagaa gaacatcaag gagtttctgc aatcctttgt gcacattgtc 1320

cagatgttca tcaatacctc c 1341

<210> 169

<211> 465

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 169

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp

20 25 30

Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp

35 40 45

Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu

50 55 60

Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr

65 70 75 80

Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly

85 90 95

Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys

100 105 110

Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

115 120 125

Ile Asn Thr Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu

130 135 140

Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys

145 150 155 160

Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp

165 170 175

Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr

180 185 190

Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe

195 200 205

Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro

210 215 220

Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu

225 230 235 240

Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn

245 250 255

Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe

260 265 270

Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr

275 280 285

Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr

290 295 300

Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser

305 310 315 320

Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp

325 330 335

Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Asn

340 345 350

Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln

355 360 365

Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro

370 375 380

Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val

385 390 395 400

Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn

405 410 415

Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr

420 425 430

Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys

435 440 445

Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr

450 455 460

Ser

465

<210> 170

<211> 1395

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 170

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccaactgg 60

gtgaacgtga tcagcgattt aaagaagatc gaggatttaa tccagagcat gcacatcgac 120

gccactctgt acactgagag cgacgtgcac cctagctgca aggtgactgc catgaagtgc 180

tttttactgg agctgcaagt tatctcttta gagagcggcg atgccagcat ccacgacact 240

gtggagaatt taatcatttt agccaacaac tctttaagca gcaacggcaa cgtgacagag 300

agcggctgca aggagtgcga ggagctggag gagaagaaca tcaaggagtt tttacagagc 360

ttcgtgcaca tcgtgcagat gttcatcaac actagcagcg gcacaaccaa cacagtcgct 420

gcctataacc tcacttggaa gagcaccaac ttcaaaacca tcctcgaatg ggaacccaaa 480

cccgttaacc aagtttacac cgtgcagatc agcaccaagt ccggcgactg gaagtccaaa 540

tgtttctata ccaccgacac cgagtgcgat ctcaccgatg agatcgtgaa agatgtgaaa 600

cagacctacc tcgcccgggt gtttagctac cccgccggca atgtggagag cactggttcc 660

gctggcgagc ctttatacga gaacagcccc gaatttaccc cttacctcga gaccaattta 720

ggacagccca ccatccaaag ctttgagcaa gttggcacaa aggtgaatgt gacagtggag 780

gacgagcgga ctttagtgcg gcggaacaac acctttctca gcctccggga tgtgttcggc 840

aaagatttaa tctacacact gtattactgg aagtcctctt cctccggcaa gaagacagct 900

aaaaccaaca caaacgagtt tttaatcgac gtggataaag gcgaaaacta ctgtttcagc 960

gtgcaagctg tgatcccctc ccggaccgtg aataggaaaa gcaccgatag ccccgttgag 1020

tgcatgggcc aagaaaaggg cgagttccgg gagaactggg tgaacgtcat cagcgattta 1080

aagaagatcg aagatttaat tcagtccatg catatcgacg ccactttata cacagaatcc 1140

gacgtgcacc cctcttgtaa ggtgaccgcc atgaaatgtt ttttactgga gctgcaagtt 1200

atctctttag agagcggaga cgctagcatc cacgacaccg tggagaattt aatcatttta 1260

gccaataact ctttatccag caacggcaac gtgacagagt ccggctgcaa ggagtgcgaa 1320

gagctggagg agaagaacat caaggagttt ctgcaatcct ttgtgcacat tgtccagatg 1380

ttcatcaata cctcc 1395

<210> 171

<211> 471

<212> DNA

<213> Chile person

<400> 171

tacttcggca aactggaatc caagctgagc gtgatccgga atttaaacga ccaagttctg 60

tttatcgatc aaggtaaccg gcctctgttc gaggacatga ccgactccga ttgccgggac 120

aatgcccccc ggaccatctt cattatctcc atgtacaagg acagccagcc ccggggcatg 180

gctgtgacaa ttagcgtgaa gtgtgagaaa atcagcactt tatcttgtga gaacaagatc 240

atctccttta aggaaatgaa cccccccgat aacatcaagg acaccaagtc cgatatcatc 300

ttcttccagc ggtccgtgcc cggtcacgat aacaagatgc agttcgaatc ctcctcctac 360

gagggctact ttttagcttg tgaaaaggag agggatttat tcaagctgat cctcaagaag 420

gaggacgagc tgggcgatcg ttccatcatg ttcaccgtcc aaaacgagga t 471

<210> 172

<211> 918

<212> DNA

<213> Chile person

<400> 172

atttgggaac tgaagaagga cgtctacgtg gtcgaactgg actggtatcc cgatgctccc 60

ggcgaaatgg tggtgctcac ttgtgacacc cccgaagaag acggcatcac ttggaccctc 120

gatcagagca gcgaggtgct gggctccgga aagaccctca caatccaagt taaggagttc 180

ggagacgctg gccaatacac atgccacaag ggaggcgagg tgctcagcca ttccttatta 240

ttattacaca agaaggaaga cggaatctgg tccaccgaca ttttaaaaga tcagaaggag 300

cccaagaata agaccttttt aaggtgtgag gccaaaaact acagcggtcg tttcacttgt 360

tggtggctga ccaccatttc caccgattta accttctccg tgaaaagcag ccggggaagc 420

tccgaccctc aaggtgtgac atgtggagcc gctaccctca gcgctgagag ggttcgtggc 480

gataacaagg aatacgagta cagcgtggag tgccaagaag atagcgcttg tcccgctgcc 540

gaagaatctt tacccattga ggtgatggtg gacgccgtgc acaaactcaa gtacgagaac 600

tacacctcct ccttctttat ccgggacatc attaagcccg atcctcctaa gaatttacag 660

ctgaagcctc tcaaaaatag ccggcaagtt gaggtctctt gggaatatcc cgacacttgg 720

agcacacccc acagctactt ctctttaacc ttttgtgtgc aagttcaagg taaaagcaag 780

cgggagaaga aagaccgggt gtttaccgac aaaaccagcg ccaccgtcat ctgtcggaag 840

aacgcctcca tcagcgtgag ggctcaagat cgttattact ccagcagctg gtccgagtgg 900

gccagcgtgc cttgttcc 918

<210> 173

<211> 591

<212> DNA

<213> Chile person

<400> 173

cgtaacctcc ccgtggctac ccccgatccc ggaatgttcc cttgtttaca ccacagccag 60

aatttactga gggccgtgag caacatgctg cagaaagcta ggcagacttt agaattttac 120

ccttgcacca gcgaggagat cgaccatgaa gatatcacca aggacaagac atccaccgtg 180

gaggcttgtt tacctctgga gctgacaaag aacgagtctt gtctcaactc tcgtgaaacc 240

agcttcatca caaatggctc ttgtttagct tcccggaaga cctcctttat gatggcttta 300

tgcctcagct ccatctacga ggatttaaag atgtaccaag tggagttcaa gaccatgaac 360

gccaagctgc tcatggaccc taaacggcag atctttttag accagaacat gctggctgtg 420

attgatgagc tgatgcaagc tttaaacttc aactccgaga ccgtccctca gaagtcctcc 480

ctcgaggagc ccgattttta caagacaaag atcaaactgt gcattttact ccacgccttt 540

aggatccggg ccgtgaccat tgaccgggtc atgagctatt taaacgccag c 591

<210> 174

<211> 490

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 174

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp Ser Gly Thr

145 150 155 160

Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe

165 170 175

Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr

180 185 190

Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr

195 200 205

Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val

210 215 220

Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val

225 230 235 240

Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu

245 250 255

Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser

260 265 270

Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg

275 280 285

Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe

290 295 300

Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser

305 310 315 320

Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val

325 330 335

Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser

340 345 350

Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly

355 360 365

Gln Glu Lys Gly Glu Phe Arg Glu Asn Trp Val Asn Val Ile Ser Asp

370 375 380

Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr

385 390 395 400

Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met

405 410 415

Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp

420 425 430

Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn

435 440 445

Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys

450 455 460

Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val

465 470 475 480

His Ile Val Gln Met Phe Ile Asn Thr Ser

485 490

<210> 175

<211> 1470

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 175

tacttcggca aactggaatc caagctgagc gtgatccgga atttaaacga ccaagttctg 60

tttatcgatc aaggtaaccg gcctctgttc gaggacatga ccgactccga ttgccgggac 120

aatgcccccc ggaccatctt cattatctcc atgtacaagg acagccagcc ccggggcatg 180

gctgtgacaa ttagcgtgaa gtgtgagaaa atcagcactt tatcttgtga gaacaagatc 240

atctccttta aggaaatgaa cccccccgat aacatcaagg acaccaagtc cgatatcatc 300

ttcttccagc ggtccgtgcc cggtcacgat aacaagatgc agttcgaatc ctcctcctac 360

gagggctact ttttagcttg tgaaaaggag agggatttat tcaagctgat cctcaagaag 420

gaggacgagc tgggcgatcg ttccatcatg ttcaccgtcc aaaacgagga tagcggcaca 480

accaacacag tcgctgccta taacctcact tggaagagca ccaacttcaa aaccatcctc 540

gaatgggaac ccaaacccgt taaccaagtt tacaccgtgc agatcagcac caagtccggc 600

gactggaagt ccaaatgttt ctataccacc gacaccgagt gcgatctcac cgatgagatc 660

gtgaaagatg tgaaacagac ctacctcgcc cgggtgttta gctaccccgc cggcaatgtg 720

gagagcactg gttccgctgg cgagccttta tacgagaaca gccccgaatt taccccttac 780

ctcgagacca atttaggaca gcccaccatc caaagctttg agcaagttgg cacaaaggtg 840

aatgtgacag tggaggacga gcggacttta gtgcggcgga acaacacctt tctcagcctc 900

cgggatgtgt tcggcaaaga tttaatctac acactgtatt actggaagtc ctcttcctcc 960

ggcaagaaga cagctaaaac caacacaaac gagtttttaa tcgacgtgga taaaggcgaa 1020

aactactgtt tcagcgtgca agctgtgatc ccctcccgga ccgtgaatag gaaaagcacc 1080

gatagccccg ttgagtgcat gggccaagaa aagggcgagt tccgggagaa ctgggtgaac 1140

gtcatcagcg atttaaagaa gatcgaagat ttaattcagt ccatgcatat cgacgccact 1200

ttatacacag aatccgacgt gcacccctct tgtaaggtga ccgccatgaa atgtttttta 1260

ctggagctgc aagttatctc tttagagagc ggagacgcta gcatccacga caccgtggag 1320

aatttaatca ttttagccaa taactcttta tccagcaacg gcaacgtgac agagtccggc 1380

tgcaaggagt gcgaagagct ggaggagaag aacatcaagg agtttctgca atcctttgtg 1440

cacattgtcc agatgttcat caatacctcc 1470

<210> 176

<211> 508

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 176

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn

20 25 30

Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe

35 40 45

Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile

50 55 60

Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val

65 70 75 80

Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn

85 90 95

Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp

100 105 110

Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp

115 120 125

Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala

130 135 140

Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp

145 150 155 160

Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp Ser

165 170 175

Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr

180 185 190

Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val

195 200 205

Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys

210 215 220

Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys

225 230 235 240

Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly

245 250 255

Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser

260 265 270

Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile

275 280 285

Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp

290 295 300

Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp

305 310 315 320

Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser

325 330 335

Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile

340 345 350

Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile

355 360 365

Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys

370 375 380

Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Asn Trp Val Asn Val Ile

385 390 395 400

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

405 410 415

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

420 425 430

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser

435 440 445

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

450 455 460

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

465 470 475 480

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

485 490 495

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

500 505

<210> 177

<211> 1524

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 177

atgaagtggg tcacatttat ctctttactg ttcctcttct ccagcgccta cagctacttc 60

ggcaaactgg aatccaagct gagcgtgatc cggaatttaa acgaccaagt tctgtttatc 120

gatcaaggta accggcctct gttcgaggac atgaccgact ccgattgccg ggacaatgcc 180

ccccggacca tcttcattat ctccatgtac aaggacagcc agccccgggg catggctgtg 240

acaattagcg tgaagtgtga gaaaatcagc actttatctt gtgagaacaa gatcatctcc 300

tttaaggaaa tgaacccccc cgataacatc aaggacacca agtccgatat catcttcttc 360

cagcggtccg tgcccggtca cgataacaag atgcagttcg aatcctcctc ctacgagggc 420

tactttttag cttgtgaaaa ggagagggat ttattcaagc tgatcctcaa gaaggaggac 480

gagctgggcg atcgttccat catgttcacc gtccaaaacg aggatagcgg cacaaccaac 540

acagtcgctg cctataacct cacttggaag agcaccaact tcaaaaccat cctcgaatgg 600

gaacccaaac ccgttaacca agtttacacc gtgcagatca gcaccaagtc cggcgactgg 660

aagtccaaat gtttctatac caccgacacc gagtgcgatc tcaccgatga gatcgtgaaa 720

gatgtgaaac agacctacct cgcccgggtg tttagctacc ccgccggcaa tgtggagagc 780

actggttccg ctggcgagcc tttatacgag aacagccccg aatttacccc ttacctcgag 840

accaatttag gacagcccac catccaaagc tttgagcaag ttggcacaaa ggtgaatgtg 900

acagtggagg acgagcggac tttagtgcgg cggaacaaca cctttctcag cctccgggat 960

gtgttcggca aagatttaat ctacacactg tattactgga agtcctcttc ctccggcaag 1020

aagacagcta aaaccaacac aaacgagttt ttaatcgacg tggataaagg cgaaaactac 1080

tgtttcagcg tgcaagctgt gatcccctcc cggaccgtga ataggaaaag caccgatagc 1140

cccgttgagt gcatgggcca agaaaagggc gagttccggg agaactgggt gaacgtcatc 1200

agcgatttaa agaagatcga agatttaatt cagtccatgc atatcgacgc cactttatac 1260

acagaatccg acgtgcaccc ctcttgtaag gtgaccgcca tgaaatgttt tttactggag 1320

ctgcaagtta tctctttaga gagcggagac gctagcatcc acgacaccgt ggagaattta 1380

atcattttag ccaataactc tttatccagc aacggcaacg tgacagagtc cggctgcaag 1440

gagtgcgaag agctggagga gaagaacatc aaggagtttc tgcaatcctt tgtgcacatt 1500

gtccagatgt tcatcaatac ctcc 1524

<210> 178

<211> 583

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 178

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

305 310 315 320

Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys

325 330 335

Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln

340 345 350

Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile

355 360 365

Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys

370 375 380

Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu

385 390 395 400

Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser

405 410 415

Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met

420 425 430

Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro

435 440 445

Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu

450 455 460

Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser

465 470 475 480

Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile

485 490 495

Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met

500 505 510

Ser Tyr Leu Asn Ala Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu

515 520 525

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

530 535 540

Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu

545 550 555 560

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

565 570 575

Pro Ser Leu Lys Cys Ile Arg

580

<210> 179

<211> 1749

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 179

atttgggaac tgaagaagga cgtctacgtg gtcgaactgg actggtatcc cgatgctccc 60

ggcgaaatgg tggtgctcac ttgtgacacc cccgaagaag acggcatcac ttggaccctc 120

gatcagagca gcgaggtgct gggctccgga aagaccctca caatccaagt taaggagttc 180

ggagacgctg gccaatacac atgccacaag ggaggcgagg tgctcagcca ttccttatta 240

ttattacaca agaaggaaga cggaatctgg tccaccgaca ttttaaaaga tcagaaggag 300

cccaagaata agaccttttt aaggtgtgag gccaaaaact acagcggtcg tttcacttgt 360

tggtggctga ccaccatttc caccgattta accttctccg tgaaaagcag ccggggaagc 420

tccgaccctc aaggtgtgac atgtggagcc gctaccctca gcgctgagag ggttcgtggc 480

gataacaagg aatacgagta cagcgtggag tgccaagaag atagcgcttg tcccgctgcc 540

gaagaatctt tacccattga ggtgatggtg gacgccgtgc acaaactcaa gtacgagaac 600

tacacctcct ccttctttat ccgggacatc attaagcccg atcctcctaa gaatttacag 660

ctgaagcctc tcaaaaatag ccggcaagtt gaggtctctt gggaatatcc cgacacttgg 720

agcacacccc acagctactt ctctttaacc ttttgtgtgc aagttcaagg taaaagcaag 780

cgggagaaga aagaccgggt gtttaccgac aaaaccagcg ccaccgtcat ctgtcggaag 840

aacgcctcca tcagcgtgag ggctcaagat cgttattact ccagcagctg gtccgagtgg 900

gccagcgtgc cttgttccgg cggtggagga tccggaggag gtggctccgg cggcggagga 960

tctcgtaacc tccccgtggc tacccccgat cccggaatgt tcccttgttt acaccacagc 1020

cagaatttac tgagggccgt gagcaacatg ctgcagaaag ctaggcagac tttagaattt 1080

tacccttgca ccagcgagga gatcgaccat gaagatatca ccaaggacaa gacatccacc 1140

gtggaggctt gtttacctct ggagctgaca aagaacgagt cttgtctcaa ctctcgtgaa 1200

accagcttca tcacaaatgg ctcttgttta gcttcccgga agacctcctt tatgatggct 1260

ttatgcctca gctccatcta cgaggattta aagatgtacc aagtggagtt caagaccatg 1320

aacgccaagc tgctcatgga ccctaaacgg cagatctttt tagaccagaa catgctggct 1380

gtgattgatg agctgatgca agctttaaac ttcaactccg agaccgtccc tcagaagtcc 1440

tccctcgagg agcccgattt ttacaagaca aagatcaaac tgtgcatttt actccacgcc 1500

tttaggatcc gggccgtgac cattgaccgg gtcatgagct atttaaacgc cagcattaca 1560

tgcccccctc ccatgagcgt ggagcacgcc gacatctggg tgaagagcta tagcctctac 1620

agccgggaga ggtatatctg taacagcggc ttcaagagga aggccggcac cagcagcctc 1680

accgagtgcg tgctgaataa ggctaccaac gtggctcact ggacaacacc ctctttaaag 1740

tgcatccgg 1749

<210> 180

<211> 601

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 180

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp

20 25 30

Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr

35 40 45

Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val

50 55 60

Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp

65 70 75 80

Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser

85 90 95

Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile

100 105 110

Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu

115 120 125

Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile

130 135 140

Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp

145 150 155 160

Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val

165 170 175

Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp

180 185 190

Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val

195 200 205

Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe

210 215 220

Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys

225 230 235 240

Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp

245 250 255

Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln

260 265 270

Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp

275 280 285

Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val

290 295 300

Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser

305 310 315 320

Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

325 330 335

Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe

340 345 350

Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met

355 360 365

Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu

370 375 380

Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu

385 390 395 400

Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser

405 410 415

Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys

420 425 430

Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu

435 440 445

Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met

450 455 460

Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile

465 470 475 480

Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln

485 490 495

Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu

500 505 510

Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg

515 520 525

Val Met Ser Tyr Leu Asn Ala Ser Ile Thr Cys Pro Pro Pro Met Ser

530 535 540

Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg

545 550 555 560

Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser

565 570 575

Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp

580 585 590

Thr Thr Pro Ser Leu Lys Cys Ile Arg

595 600

<210> 181

<211> 1803

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 181

atgaaatggg tgacctttat ttctttactg ttcctcttta gcagcgccta ctccatttgg 60

gaactgaaga aggacgtcta cgtggtcgaa ctggactggt atcccgatgc tcccggcgaa 120

atggtggtgc tcacttgtga cacccccgaa gaagacggca tcacttggac cctcgatcag 180

agcagcgagg tgctgggctc cggaaagacc ctcacaatcc aagttaagga gttcggagac 240

gctggccaat acacatgcca caagggaggc gaggtgctca gccattcctt attattatta 300

cacaagaagg aagacggaat ctggtccacc gacattttaa aagatcagaa ggagcccaag 360

aataagacct ttttaaggtg tgaggccaaa aactacagcg gtcgtttcac ttgttggtgg 420

ctgaccacca tttccaccga tttaaccttc tccgtgaaaa gcagccgggg aagctccgac 480

cctcaaggtg tgacatgtgg agccgctacc ctcagcgctg agagggttcg tggcgataac 540

aaggaatacg agtacagcgt ggagtgccaa gaagatagcg cttgtcccgc tgccgaagaa 600

tctttaccca ttgaggtgat ggtggacgcc gtgcacaaac tcaagtacga gaactacacc 660

tcctccttct ttatccggga catcattaag cccgatcctc ctaagaattt acagctgaag 720

cctctcaaaa atagccggca agttgaggtc tcttgggaat atcccgacac ttggagcaca 780

ccccacagct acttctcttt aaccttttgt gtgcaagttc aaggtaaaag caagcgggag 840

aagaaagacc gggtgtttac cgacaaaacc agcgccaccg tcatctgtcg gaagaacgcc 900

tccatcagcg tgagggctca agatcgttat tactccagca gctggtccga gtgggccagc 960

gtgccttgtt ccggcggtgg aggatccgga ggaggtggct ccggcggcgg aggatctcgt 1020

aacctccccg tggctacccc cgatcccgga atgttccctt gtttacacca cagccagaat 1080

ttactgaggg ccgtgagcaa catgctgcag aaagctaggc agactttaga attttaccct 1140

tgcaccagcg aggagatcga ccatgaagat atcaccaagg acaagacatc caccgtggag 1200

gcttgtttac ctctggagct gacaaagaac gagtcttgtc tcaactctcg tgaaaccagc 1260

ttcatcacaa atggctcttg tttagcttcc cggaagacct cctttatgat ggctttatgc 1320

ctcagctcca tctacgagga tttaaagatg taccaagtgg agttcaagac catgaacgcc 1380

aagctgctca tggaccctaa acggcagatc tttttagacc agaacatgct ggctgtgatt 1440

gatgagctga tgcaagcttt aaacttcaac tccgagaccg tccctcagaa gtcctccctc 1500

gaggagcccg atttttacaa gacaaagatc aaactgtgca ttttactcca cgcctttagg 1560

atccgggccg tgaccattga ccgggtcatg agctatttaa acgccagcat tacatgcccc 1620

cctcccatga gcgtggagca cgccgacatc tgggtgaaga gctatagcct ctacagccgg 1680

gagaggtata tctgtaacag cggcttcaag aggaaggccg gcaccagcag cctcaccgag 1740

tgcgtgctga ataaggctac caacgtggct cactggacaa caccctcttt aaagtgcatc 1800

cgg 1803

<210> 182

<211> 399

<212> DNA

<213> Chile person

<400> 182

cagggccagg acaggcacat gatccggatg aggcagctca tcgacatcgt cgaccagctg 60

aagaactacg tgaacgacct ggtgcccgag tttctgcctg cccccgagga cgtggagacc 120

aactgcgagt ggtccgcctt ctcctgcttt cagaaggccc agctgaagtc cgccaacacc 180

ggcaacaacg agcggatcat caacgtgagc atcaagaagc tgaagcggaa gcctccctcc 240

acaaacgccg gcaggaggca gaagcacagg ctgacctgcc ccagctgtga ctcctacgag 300

aagaagcccc ccaaggagtt cctggagagg ttcaagtccc tgctgcagaa gatgatccat 360

cagcacctgt cctccaggac ccacggctcc gaggactcc 399

<210> 183

<211> 136

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 183

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 184

<211> 136

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 184

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 185

<211> 408

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 185

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcacgatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgat 408

<210> 186

<211> 408

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 186

attcctcccc acgtgcagaa gagcgtgaat aatgacatga tcgtgaccga taacaatggc 60

gccgtgaaat ttccccagct gtgcaaattc tgcgatgtga ggttttccac ctgcgacaac 120

cagaagtcct gtatgagcaa ctgcacaatc acctccatct gtgagaagcc tcaggaggtg 180

tgcgtggctg tctggcggaa gaatgacgag aatatcaccc tggaaaccgt ctgccacgat 240

cccaagctgc cctaccacga tttcatcctg gaagacgccg ccagccctaa gtgcatcatg 300

aaagagaaaa agaagcctgg cgagaccttt ttcatgtgct cctgcagcag cgacgaatgc 360

aacgacaata tcatctttag cgaggaatac aataccagca accccgac 408

<210> 187

<211> 861

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 187

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcacgatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgcaca 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga c 861

<210> 188

<211> 287

<212> PRT

<213> Chile person

<400> 188

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val

145 150 155 160

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

165 170 175

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

180 185 190

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

195 200 205

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

210 215 220

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

225 230 235 240

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

245 250 255

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

260 265 270

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

275 280 285

<210> 189

<211> 466

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 189

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn

130 135 140

Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro

145 150 155 160

Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly

165 170 175

Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu

180 185 190

Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val

195 200 205

Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu

210 215 220

Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn

225 230 235 240

Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val

245 250 255

Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr

260 265 270

Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu

275 280 285

Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn

290 295 300

Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe

305 310 315 320

Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr

325 330 335

Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu

340 345 350

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

355 360 365

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

370 375 380

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

385 390 395 400

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

405 410 415

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

420 425 430

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

435 440 445

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

450 455 460

Thr Ser

465

<210> 190

<211> 1398

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 190

cagggccagg acaggcacat gatccggatg aggcagctca tcgacatcgt cgaccagctg 60

aagaactacg tgaacgacct ggtgcccgag tttctgcctg cccccgagga cgtggagacc 120

aactgcgagt ggtccgcctt ctcctgcttt cagaaggccc agctgaagtc cgccaacacc 180

ggcaacaacg agcggatcat caacgtgagc atcaagaagc tgaagcggaa gcctccctcc 240

acaaacgccg gcaggaggca gaagcacagg ctgacctgcc ccagctgtga ctcctacgag 300

aagaagcccc ccaaggagtt cctggagagg ttcaagtccc tgctgcagaa gatgatccat 360

cagcacctgt cctccaggac ccacggctcc gaggactcct ccggcaccac caataccgtg 420

gccgcttata acctcacatg gaagagcacc aacttcaaga caattctgga atgggaaccc 480

aagcccgtca atcaagttta caccgtgcag atctccacca aatccggaga ctggaagagc 540

aagtgcttct acacaacaga caccgagtgt gatttaaccg acgaaatcgt caaggacgtc 600

aagcaaacct atctggctcg ggtcttttcc taccccgctg gcaatgtcga gtccaccggc 660

tccgctggcg agcctctcta cgagaattcc cccgaattca ccccttattt agagaccaat 720

ttaggccagc ctaccatcca gagcttcgag caagttggca ccaaggtgaa cgtcaccgtc 780

gaggatgaaa ggactttagt gcggcggaat aacacatttt tatccctccg ggatgtgttc 840

ggcaaagacc tcatctacac actgtactat tggaagtcca gctcctccgg caaaaagacc 900

gctaagacca acaccaacga gtttttaatt gacgtggaca aaggcgagaa ctactgcttc 960

agcgtgcaag ccgtgatccc ttctcgtacc gtcaaccgga agagcacaga ttcccccgtt 1020

gagtgcatgg gccaagaaaa gggcgagttc cgggagaact gggtgaacgt catcagcgat 1080

ttaaagaaga tcgaagattt aattcagtcc atgcatatcg acgccacttt atacacagaa 1140

tccgacgtgc acccctcttg taaggtgacc gccatgaaat gttttttact ggagctgcaa 1200

gttatctctt tagagagcgg agacgctagc atccacgaca ccgtggagaa tttaatcatt 1260

ttagccaata actctttatc cagcaacggc aacgtgacag agtccggctg caaggagtgc 1320

gaagagctgg aggagaagaa catcaaggag tttctgcaat cctttgtgca cattgtccag 1380

atgttcatca atacctcc 1398

<210> 191

<211> 484

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 191

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

20 25 30

Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

35 40 45

Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

50 55 60

Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn

65 70 75 80

Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

85 90 95

Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

100 105 110

Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

115 120 125

Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg

130 135 140

Thr His Gly Ser Glu Asp Ser Ser Gly Thr Thr Asn Thr Val Ala Ala

145 150 155 160

Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp

165 170 175

Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys

180 185 190

Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys

195 200 205

Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala

210 215 220

Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala

225 230 235 240

Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu

245 250 255

Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr

260 265 270

Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn

275 280 285

Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr

290 295 300

Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys

305 310 315 320

Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr

325 330 335

Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys

340 345 350

Ser Thr Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe

355 360 365

Arg Glu Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp

370 375 380

Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp

385 390 395 400

Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu

405 410 415

Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr

420 425 430

Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly

435 440 445

Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys

450 455 460

Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

465 470 475 480

Ile Asn Thr Ser

<210> 192

<211> 1452

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 192

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctcccagggc 60

caggacaggc acatgatccg gatgaggcag ctcatcgaca tcgtcgacca gctgaagaac 120

tacgtgaacg acctggtgcc cgagtttctg cctgcccccg aggacgtgga gaccaactgc 180

gagtggtccg ccttctcctg ctttcagaag gcccagctga agtccgccaa caccggcaac 240

aacgagcgga tcatcaacgt gagcatcaag aagctgaagc ggaagcctcc ctccacaaac 300

gccggcagga ggcagaagca caggctgacc tgccccagct gtgactccta cgagaagaag 360

ccccccaagg agttcctgga gaggttcaag tccctgctgc agaagatgat ccatcagcac 420

ctgtcctcca ggacccacgg ctccgaggac tcctccggca ccaccaatac cgtggccgct 480

tataacctca catggaagag caccaacttc aagacaattc tggaatggga acccaagccc 540

gtcaatcaag tttacaccgt gcagatctcc accaaatccg gagactggaa gagcaagtgc 600

ttctacacaa cagacaccga gtgtgattta accgacgaaa tcgtcaagga cgtcaagcaa 660

acctatctgg ctcgggtctt ttcctacccc gctggcaatg tcgagtccac cggctccgct 720

ggcgagcctc tctacgagaa ttcccccgaa ttcacccctt atttagagac caatttaggc 780

cagcctacca tccagagctt cgagcaagtt ggcaccaagg tgaacgtcac cgtcgaggat 840

gaaaggactt tagtgcggcg gaataacaca tttttatccc tccgggatgt gttcggcaaa 900

gacctcatct acacactgta ctattggaag tccagctcct ccggcaaaaa gaccgctaag 960

accaacacca acgagttttt aattgacgtg gacaaaggcg agaactactg cttcagcgtg 1020

caagccgtga tcccttctcg taccgtcaac cggaagagca cagattcccc cgttgagtgc 1080

atgggccaag aaaagggcga gttccgggag aactgggtga acgtcatcag cgatttaaag 1140

aagatcgaag atttaattca gtccatgcat atcgacgcca ctttatacac agaatccgac 1200

gtgcacccct cttgtaaggt gaccgccatg aaatgttttt tactggagct gcaagttatc 1260

tctttagaga gcggagacgc tagcatccac gacaccgtgg agaatttaat cattttagcc 1320

aataactctt tatccagcaa cggcaacgtg acagagtccg gctgcaagga gtgcgaagag 1380

ctggaggaga agaacatcaa ggagtttctg caatcctttg tgcacattgt ccagatgttc 1440

atcaatacct cc 1452

<210> 193

<211> 352

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 193

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val

145 150 155 160

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

165 170 175

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

180 185 190

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

195 200 205

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

210 215 220

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

225 230 235 240

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

245 250 255

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

260 265 270

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile

275 280 285

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

290 295 300

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

305 310 315 320

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

325 330 335

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

340 345 350

<210> 194

<211> 1056

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 194

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcacgatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgcaca 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga catcacgtgt cctcctccta tgtccgtgga acacgcagac 900

atctgggtca agagctacag cttgtactcc agggagcggt acatttgtaa ctctggtttc 960

aagcgtaaag ccggcacgtc cagcctgacg gagtgcgtgt tgaacaaggc cacgaatgtc 1020

gcccactgga caacccccag tctcaaatgt attaga 1056

<210> 195

<211> 370

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 195

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

20 25 30

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

35 40 45

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

50 55 60

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

65 70 75 80

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

85 90 95

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

100 105 110

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

115 120 125

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

130 135 140

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys

165 170 175

Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys

180 185 190

Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp

195 200 205

Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu

210 215 220

Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn

225 230 235 240

Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp

245 250 255

Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys

260 265 270

Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu

275 280 285

Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro

290 295 300

Asp Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

305 310 315 320

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

325 330 335

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

340 345 350

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

355 360 365

Ile Arg

370

<210> 196

<211> 1110

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 196

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccatcccc 60

ccccatgtgc aaaagagcgt gaacaacgat atgatcgtga ccgacaacaa cggcgccgtg 120

aagtttcccc agctctgcaa gttctgcgat gtcaggttca gcacctgcga taatcagaag 180

tcctgcatgt ccaactgcac gatcacctcc atctgcgaga agccccaaga agtgtgcgtg 240

gccgtgtggc ggaaaaatga cgagaacatc accctggaga ccgtgtgtca cgaccccaag 300

ctcccttatc acgacttcat tctggaggac gctgcctccc ccaaatgcat catgaaggag 360

aagaagaagc ccggagagac cttctttatg tgttcctgta gcagcgacga gtgtaacgac 420

aacatcatct tcagcgaaga gtacaacacc agcaaccctg atggaggtgg cggatccgga 480

ggtggaggtt ctggtggagg tgggagtatt cctccccacg tgcagaagag cgtgaataat 540

gacatgatcg tgaccgataa caatggcgcc gtgaaatttc cccagctgtg caaattctgc 600

gatgtgaggt tttccacctg cgacaaccag aagtcctgta tgagcaactg cacaatcacc 660

tccatctgtg agaagcctca ggaggtgtgc gtggctgtct ggcggaagaa tgacgagaat 720

atcaccctgg aaaccgtctg ccacgatccc aagctgccct accacgattt catcctggaa 780

gacgccgcca gccctaagtg catcatgaaa gagaaaaaga agcctggcga gacctttttc 840

atgtgctcct gcagcagcga cgaatgcaac gacaatatca tctttagcga ggaatacaat 900

accagcaacc ccgacatcac gtgtcctcct cctatgtccg tggaacacgc agacatctgg 960

gtcaagagct acagcttgta ctccagggag cggtacattt gtaactctgg tttcaagcgt 1020

aaagccggca cgtccagcct gacggagtgc gtgttgaaca aggccacgaa tgtcgcccac 1080

tggacaaccc ccagtctcaa atgtattaga 1110

<210> 197

<211> 399

<212> DNA

<213> Chile person

<400> 197

caaggtcaag atcgccacat gattagaatg cgtcaactta tagatattgt tgatcagctg 60

aaaaattatg tgaatgactt ggtccctgaa tttctgccag ctccagaaga tgtagagaca 120

aactgtgagt ggtcagcttt ttcctgtttt cagaaggccc aactaaagtc agcaaataca 180

ggaaacaatg aaaggataat caatgtatca attaaaaagc tgaagaggaa accaccttcc 240

acaaatgcag ggagaagaca gaaacacaga ctaacatgcc cttcatgtga ttcttatgag 300

aaaaaaccac ccaaagaatt cctagaaaga ttcaaatcac ttctccaaaa gatgattcat 360

cagcatctgt cctctagaac acacggaagt gaagattcc 399

<210> 198

<211> 456

<212> DNA

<213> Chile person

<400> 198

gattgtgata ttgaaggtaa agatggcaaa caatatgaga gtgttctaat ggtcagcatc 60

gatcaattat tggacagcat gaaagaaatt ggtagcaatt gcctgaataa tgaatttaac 120

ttttttaaaa gacatatctg tgatgctaat aaggaaggta tgtttttatt ccgtgctgct 180

cgcaagttga ggcaatttct taaaatgaat agcactggtg attttgatct ccacttatta 240

aaagtttcag aaggcacaac aatactgttg aactgcactg gccaggttaa aggaagaaaa 300

ccagctgccc tgggtgaagc ccaaccaaca aagagtttgg aagaaaataa atctttaaag 360

gaacagaaaa aactgaatga cttgtgtttc ctaaagagac tattacaaga gataaaaact 420

tgttggaata aaattttgat gggcactaaa gaacac 456

<210> 199

<211> 466

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 199

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn

130 135 140

Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro

145 150 155 160

Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly

165 170 175

Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu

180 185 190

Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val

195 200 205

Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu

210 215 220

Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn

225 230 235 240

Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val

245 250 255

Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr

260 265 270

Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu

275 280 285

Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn

290 295 300

Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe

305 310 315 320

Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr

325 330 335

Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu

340 345 350

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

355 360 365

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

370 375 380

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

385 390 395 400

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

405 410 415

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

420 425 430

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

435 440 445

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

450 455 460

Thr Ser

465

<210> 200

<211> 1398

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 200

caaggtcaag atcgccacat gattagaatg cgtcaactta tagatattgt tgatcagctg 60

aaaaattatg tgaatgactt ggtccctgaa tttctgccag ctccagaaga tgtagagaca 120

aactgtgagt ggtcagcttt ttcctgtttt cagaaggccc aactaaagtc agcaaataca 180

ggaaacaatg aaaggataat caatgtatca attaaaaagc tgaagaggaa accaccttcc 240

acaaatgcag ggagaagaca gaaacacaga ctaacatgcc cttcatgtga ttcttatgag 300

aaaaaaccac ccaaagaatt cctagaaaga ttcaaatcac ttctccaaaa gatgattcat 360

cagcatctgt cctctagaac acacggaagt gaagattcct caggcactac aaatactgtg 420

gcagcatata atttaacttg gaaatcaact aatttcaaga caattttgga gtgggaaccc 480

aaacccgtca atcaagtcta cactgttcaa ataagcacta agtcaggaga ttggaaaagc 540

aaatgctttt acacaacaga cacagagtgt gacctcaccg acgagattgt gaaggatgtg 600

aagcagacgt acttggcacg ggtcttctcc tacccggcag ggaatgtgga gagcaccggt 660

tctgctgggg agcctctgta tgagaactcc ccagagttca caccttacct ggagacaaac 720

ctcggacagc caacaattca gagttttgaa caggtgggaa caaaagtgaa tgtgaccgta 780

gaagatgaac ggactttagt cagaaggaac aacactttcc taagcctccg ggatgttttt 840

ggcaaggact taatttatac actttattat tggaaatctt caagttcagg aaagaaaaca 900

gccaaaacaa acactaatga gtttttgatt gatgtggata aaggagaaaa ctactgtttc 960

agtgttcaag cagtgattcc ctcccgaaca gttaaccgga agagtacaga cagcccggta 1020

gagtgtatgg gccaggagaa aggggaattc agagaaaact gggtgaacgt catcagcgat 1080

ttaaagaaga tcgaagattt aattcagtcc atgcatatcg acgccacttt atacacagaa 1140

tccgacgtgc acccctcttg taaggtgacc gccatgaaat gttttttact ggagctgcaa 1200

gttatctctt tagagagcgg agacgctagc atccacgaca ccgtggagaa tttaatcatt 1260

ttagccaata actctttatc cagcaacggc aacgtgacag agtccggctg caaggagtgc 1320

gaagagctgg aggagaagaa catcaaggag tttctgcaat cctttgtgca cattgtccag 1380

atgttcatca atacctcc 1398

<210> 201

<211> 483

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 201

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp

20 25 30

Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe

35 40 45

Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe

50 55 60

Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn

65 70 75 80

Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro

85 90 95

Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser

100 105 110

Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe

115 120 125

Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr

130 135 140

His Gly Ser Glu Asp Ser Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr

145 150 155 160

Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu

165 170 175

Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser

180 185 190

Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp

195 200 205

Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg

210 215 220

Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly

225 230 235 240

Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr

245 250 255

Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys

260 265 270

Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn

275 280 285

Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr

290 295 300

Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr

305 310 315 320

Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys

325 330 335

Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser

340 345 350

Thr Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg

355 360 365

Glu Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

370 375 380

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

385 390 395 400

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

405 410 415

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

420 425 430

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

435 440 445

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

450 455 460

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

465 470 475 480

Asn Thr Ser

<210> 202

<211> 1449

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 202

atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc ccaaggtcaa 60

gatcgccaca tgattagaat gcgtcaactt atagatattg ttgatcagct gaaaaattat 120

gtgaatgact tggtccctga atttctgcca gctccagaag atgtagagac aaactgtgag 180

tggtcagctt tttcctgttt tcagaaggcc caactaaagt cagcaaatac aggaaacaat 240

gaaaggataa tcaatgtatc aattaaaaag ctgaagagga aaccaccttc cacaaatgca 300

gggagaagac agaaacacag actaacatgc ccttcatgtg attcttatga gaaaaaacca 360

cccaaagaat tcctagaaag attcaaatca cttctccaaa agatgattca tcagcatctg 420

tcctctagaa cacacggaag tgaagattcc tcaggcacta caaatactgt ggcagcatat 480

aatttaactt ggaaatcaac taatttcaag acaattttgg agtgggaacc caaacccgtc 540

aatcaagtct acactgttca aataagcact aagtcaggag attggaaaag caaatgcttt 600

tacacaacag acacagagtg tgacctcacc gacgagattg tgaaggatgt gaagcagacg 660

tacttggcac gggtcttctc ctacccggca gggaatgtgg agagcaccgg ttctgctggg 720

gagcctctgt atgagaactc cccagagttc acaccttacc tggagacaaa cctcggacag 780

ccaacaattc agagttttga acaggtggga acaaaagtga atgtgaccgt agaagatgaa 840

cggactttag tcagaaggaa caacactttc ctaagcctcc gggatgtttt tggcaaggac 900

ttaatttata cactttatta ttggaaatct tcaagttcag gaaagaaaac agccaaaaca 960

aacactaatg agtttttgat tgatgtggat aaaggagaaa actactgttt cagtgttcaa 1020

gcagtgattc cctcccgaac agttaaccgg aagagtacag acagcccggt agagtgtatg 1080

ggccaggaga aaggggaatt cagagaaaac tgggtgaacg tcatcagcga tttaaagaag 1140

atcgaagatt taattcagtc catgcatatc gacgccactt tatacacaga atccgacgtg 1200

cacccctctt gtaaggtgac cgccatgaaa tgttttttac tggagctgca agttatctct 1260

ttagagagcg gagacgctag catccacgac accgtggaga atttaatcat tttagccaat 1320

aactctttat ccagcaacgg caacgtgaca gagtccggct gcaaggagtg cgaagagctg 1380

gaggagaaga acatcaagga gtttctgcaa tcctttgtgc acattgtcca gatgttcatc 1440

aatacctcc 1449

<210> 203

<211> 217

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 203

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His Ile Thr Cys Pro Pro Pro Met Ser

145 150 155 160

Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg

165 170 175

Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser

180 185 190

Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp

195 200 205

Thr Thr Pro Ser Leu Lys Cys Ile Arg

210 215

<210> 204

<211> 651

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 204

gattgtgata ttgaaggtaa agatggcaaa caatatgaga gtgttctaat ggtcagcatc 60

gatcaattat tggacagcat gaaagaaatt ggtagcaatt gcctgaataa tgaatttaac 120

ttttttaaaa gacatatctg tgatgctaat aaggaaggta tgtttttatt ccgtgctgct 180

cgcaagttga ggcaatttct taaaatgaat agcactggtg attttgatct ccacttatta 240

aaagtttcag aaggcacaac aatactgttg aactgcactg gccaggttaa aggaagaaaa 300

ccagctgccc tgggtgaagc ccaaccaaca aagagtttgg aagaaaataa atctttaaag 360

gaacagaaaa aactgaatga cttgtgtttc ctaaagagac tattacaaga gataaaaact 420

tgttggaata aaattttgat gggcactaaa gaacacatca cgtgccctcc ccccatgtcc 480

gtggaacacg cagacatctg ggtcaagagc tacagcttgt actccaggga gcggtacatt 540

tgtaactctg gtttcaagcg taaagccggc acgtccagcc tgacggagtg cgtgttgaac 600

aaggccacga atgtcgccca ctggacaacc cccagtctca aatgcattag a 651

<210> 205

<211> 234

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 205

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val

20 25 30

Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly

35 40 45

Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys

50 55 60

Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu

65 70 75 80

Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu

85 90 95

Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln

100 105 110

Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys

115 120 125

Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp

130 135 140

Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn

145 150 155 160

Lys Ile Leu Met Gly Thr Lys Glu His Ile Thr Cys Pro Pro Pro Met

165 170 175

Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser

180 185 190

Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr

195 200 205

Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His

210 215 220

Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

225 230

<210> 206

<211> 702

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 206

atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc cgattgtgat 60

attgaaggta aagatggcaa acaatatgag agtgttctaa tggtcagcat cgatcaatta 120

ttggacagca tgaaagaaat tggtagcaat tgcctgaata atgaatttaa cttttttaaa 180

agacatatct gtgatgctaa taaggaaggt atgtttttat tccgtgctgc tcgcaagttg 240

aggcaatttc ttaaaatgaa tagcactggt gattttgatc tccacttatt aaaagtttca 300

gaaggcacaa caatactgtt gaactgcact ggccaggtta aaggaagaaa accagctgcc 360

ctgggtgaag cccaaccaac aaagagtttg gaagaaaata aatctttaaa ggaacagaaa 420

aaactgaatg acttgtgttt cctaaagaga ctattacaag agataaaaac ttgttggaat 480

aaaattttga tgggcactaa agaacacatc acgtgccctc cccccatgtc cgtggaacac 540

gcagacatct gggtcaagag ctacagcttg tactccaggg agcggtacat ttgtaactct 600

ggtttcaagc gtaaagccgg cacgtccagc ctgacggagt gcgtgttgaa caaggccacg 660

aatgtcgccc actggacaac ccccagtctc aaatgcatta ga 702

<210> 207

<211> 485

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 207

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His Ser Gly Thr Thr Asn Thr Val Ala

145 150 155 160

Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu

165 170 175

Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr

180 185 190

Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu

195 200 205

Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu

210 215 220

Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser

225 230 235 240

Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu

245 250 255

Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly

260 265 270

Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg

275 280 285

Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile

290 295 300

Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala

305 310 315 320

Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn

325 330 335

Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg

340 345 350

Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu

355 360 365

Phe Arg Glu Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

370 375 380

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

385 390 395 400

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

405 410 415

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

420 425 430

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

435 440 445

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

450 455 460

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

465 470 475 480

Phe Ile Asn Thr Ser

485

<210> 208

<211> 1455

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 208

gattgcgaca tcgagggcaa ggacggcaag cagtacgaga gcgtgctgat ggtgtccatc 60

gaccagctgc tggacagcat gaaggagatc ggctccaact gcctcaacaa cgagttcaac 120

ttcttcaagc ggcacatctg cgacgccaac aaggagggca tgttcctgtt cagggccgcc 180

aggaaactgc ggcagttcct gaagatgaac tccaccggcg acttcgacct gcacctgctg 240

aaggtgtccg agggcaccac catcctgctg aactgcaccg gacaggtgaa gggccggaaa 300

cctgctgctc tgggagaggc ccaacccacc aagagcctgg aggagaacaa gtccctgaag 360

gagcagaaga agctgaacga cctgtgcttc ctgaagaggc tgctgcagga gatcaagacc 420

tgctggaaca agatcctgat gggcaccaag gagcatagcg gcacaaccaa cacagtcgct 480

gcctataacc tcacttggaa gagcaccaac ttcaaaacca tcctcgaatg ggaacccaaa 540

cccgttaacc aagtttacac cgtgcagatc agcaccaagt ccggcgactg gaagtccaaa 600

tgtttctata ccaccgacac cgagtgcgat ctcaccgatg agatcgtgaa agatgtgaaa 660

cagacctacc tcgcccgggt gtttagctac cccgccggca atgtggagag cactggttcc 720

gctggcgagc ctttatacga gaacagcccc gaatttaccc cttacctcga gaccaattta 780

ggacagccca ccatccaaag ctttgagcaa gttggcacaa aggtgaatgt gacagtggag 840

gacgagcgga ctttagtgcg gcggaacaac acctttctca gcctccggga tgtgttcggc 900

aaagatttaa tctacacact gtattactgg aagtcctctt cctccggcaa gaagacagct 960

aaaaccaaca caaacgagtt tttaatcgac gtggataaag gcgaaaacta ctgtttcagc 1020

gtgcaagctg tgatcccctc ccggaccgtg aataggaaaa gcaccgatag ccccgttgag 1080

tgcatgggcc aagaaaaggg cgagttccgg gagaactggg tgaacgtcat cagcgattta 1140

aagaagatcg aagatttaat tcagtccatg catatcgacg ccactttata cacagaatcc 1200

gacgtgcacc cctcttgtaa ggtgaccgcc atgaaatgtt ttttactgga gctgcaagtt 1260

atctctttag agagcggaga cgctagcatc cacgacaccg tggagaattt aatcatttta 1320

gccaataact ctttatccag caacggcaac gtgacagagt ccggctgcaa ggagtgcgaa 1380

gagctggagg agaagaacat caaggagttt ctgcaatcct ttgtgcacat tgtccagatg 1440

ttcatcaata cctcc 1455

<210> 209

<211> 503

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 209

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser

20 25 30

Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile

35 40 45

Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile

50 55 60

Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys

65 70 75 80

Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His

85 90 95

Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly

100 105 110

Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr

115 120 125

Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn

130 135 140

Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp

145 150 155 160

Asn Lys Ile Leu Met Gly Thr Lys Glu His Ser Gly Thr Thr Asn Thr

165 170 175

Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile

180 185 190

Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile

195 200 205

Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp

210 215 220

Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr

225 230 235 240

Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr

245 250 255

Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro

260 265 270

Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln

275 280 285

Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val

290 295 300

Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp

305 310 315 320

Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys

325 330 335

Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly

340 345 350

Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val

355 360 365

Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys

370 375 380

Gly Glu Phe Arg Glu Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys

385 390 395 400

Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr

405 410 415

Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe

420 425 430

Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile

435 440 445

His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser

450 455 460

Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu

465 470 475 480

Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val

485 490 495

Gln Met Phe Ile Asn Thr Ser

500

<210> 210

<211> 1509

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 210

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccgattgc 60

gacatcgagg gcaaggacgg caagcagtac gagagcgtgc tgatggtgtc catcgaccag 120

ctgctggaca gcatgaagga gatcggctcc aactgcctca acaacgagtt caacttcttc 180

aagcggcaca tctgcgacgc caacaaggag ggcatgttcc tgttcagggc cgccaggaaa 240

ctgcggcagt tcctgaagat gaactccacc ggcgacttcg acctgcacct gctgaaggtg 300

tccgagggca ccaccatcct gctgaactgc accggacagg tgaagggccg gaaacctgct 360

gctctgggag aggcccaacc caccaagagc ctggaggaga acaagtccct gaaggagcag 420

aagaagctga acgacctgtg cttcctgaag aggctgctgc aggagatcaa gacctgctgg 480

aacaagatcc tgatgggcac caaggagcat agcggcacaa ccaacacagt cgctgcctat 540

aacctcactt ggaagagcac caacttcaaa accatcctcg aatgggaacc caaacccgtt 600

aaccaagttt acaccgtgca gatcagcacc aagtccggcg actggaagtc caaatgtttc 660

tataccaccg acaccgagtg cgatctcacc gatgagatcg tgaaagatgt gaaacagacc 720

tacctcgccc gggtgtttag ctaccccgcc ggcaatgtgg agagcactgg ttccgctggc 780

gagcctttat acgagaacag ccccgaattt accccttacc tcgagaccaa tttaggacag 840

cccaccatcc aaagctttga gcaagttggc acaaaggtga atgtgacagt ggaggacgag 900

cggactttag tgcggcggaa caacaccttt ctcagcctcc gggatgtgtt cggcaaagat 960

ttaatctaca cactgtatta ctggaagtcc tcttcctccg gcaagaagac agctaaaacc 1020

aacacaaacg agtttttaat cgacgtggat aaaggcgaaa actactgttt cagcgtgcaa 1080

gctgtgatcc cctcccggac cgtgaatagg aaaagcaccg atagccccgt tgagtgcatg 1140

ggccaagaaa agggcgagtt ccgggagaac tgggtgaacg tcatcagcga tttaaagaag 1200

atcgaagatt taattcagtc catgcatatc gacgccactt tatacacaga atccgacgtg 1260

cacccctctt gtaaggtgac cgccatgaaa tgttttttac tggagctgca agttatctct 1320

ttagagagcg gagacgctag catccacgac accgtggaga atttaatcat tttagccaat 1380

aactctttat ccagcaacgg caacgtgaca gagtccggct gcaaggagtg cgaagagctg 1440

gaggagaaga acatcaagga gtttctgcaa tcctttgtgc acattgtcca gatgttcatc 1500

aatacctcc 1509

<210> 211

<211> 198

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 211

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His

130 135 140

Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr

145 150 155 160

Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr

165 170 175

Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro

180 185 190

Ser Leu Lys Cys Ile Arg

195

<210> 212

<211> 594

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 212

cagggccagg acaggcacat gatccggatg aggcagctca tcgacatcgt cgaccagctg 60

aagaactacg tgaacgacct ggtgcccgag tttctgcctg cccccgagga cgtggagacc 120

aactgcgagt ggtccgcctt ctcctgcttt cagaaggccc agctgaagtc cgccaacacc 180

ggcaacaacg agcggatcat caacgtgagc atcaagaagc tgaagcggaa gcctccctcc 240

acaaacgccg gcaggaggca gaagcacagg ctgacctgcc ccagctgtga ctcctacgag 300

aagaagcccc ccaaggagtt cctggagagg ttcaagtccc tgctgcagaa gatgatccat 360

cagcacctgt cctccaggac ccacggctcc gaggactcca ttacatgccc ccctcccatg 420

agcgtggagc acgccgacat ctgggtgaag agctatagcc tctacagccg ggagaggtat 480

atctgtaaca gcggcttcaa gaggaaggcc ggcaccagca gcctcaccga gtgcgtgctg 540

aataaggcta ccaacgtggc tcactggaca acaccctctt taaagtgcat ccgg 594

<210> 213

<211> 216

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 213

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

20 25 30

Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

35 40 45

Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

50 55 60

Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn

65 70 75 80

Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

85 90 95

Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

100 105 110

Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

115 120 125

Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg

130 135 140

Thr His Gly Ser Glu Asp Ser Ile Thr Cys Pro Pro Pro Met Ser Val

145 150 155 160

Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu

165 170 175

Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser

180 185 190

Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr

195 200 205

Thr Pro Ser Leu Lys Cys Ile Arg

210 215

<210> 214

<211> 648

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 214

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctcccagggc 60

caggacaggc acatgatccg gatgaggcag ctcatcgaca tcgtcgacca gctgaagaac 120

tacgtgaacg acctggtgcc cgagtttctg cctgcccccg aggacgtgga gaccaactgc 180

gagtggtccg ccttctcctg ctttcagaag gcccagctga agtccgccaa caccggcaac 240

aacgagcgga tcatcaacgt gagcatcaag aagctgaagc ggaagcctcc ctccacaaac 300

gccggcagga ggcagaagca caggctgacc tgccccagct gtgactccta cgagaagaag 360

ccccccaagg agttcctgga gaggttcaag tccctgctgc agaagatgat ccatcagcac 420

ctgtcctcca ggacccacgg ctccgaggac tccattacat gcccccctcc catgagcgtg 480

gagcacgccg acatctgggt gaagagctat agcctctaca gccgggagag gtatatctgt 540

aacagcggct tcaagaggaa ggccggcacc agcagcctca ccgagtgcgt gctgaataag 600

gctaccaacg tggctcactg gacaacaccc tctttaaagt gcatccgg 648

<210> 215

<211> 108

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 215

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr

1 5 10 15

Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly

35 40 45

Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser

50 55 60

Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Gly His

100 105

<210> 216

<211> 324

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 216

tccgagctga cccaggaccc tgctgtgtcc gtggctctgg gccagaccgt gaggatcacc 60

tgccagggcg actccctgag gtcctactac gcctcctggt accagcagaa gcccggccag 120

gctcctgtgc tggtgatcta cggcaagaac aacaggccct ccggcatccc tgacaggttc 180

tccggatcct cctccggcaa caccgcctcc ctgaccatca caggcgctca ggccgaggac 240

gaggctgact actactgcaa ctccagggac tcctccggca accatgtggt gttcggcggc 300

ggcaccaagc tgaccgtggg ccat 324

<210> 217

<211> 117

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 217

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Arg Ser Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Arg

115

<210> 218

<211> 351

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 218

gaggtgcagc tggtggagtc cggaggagga gtggtgaggc ctggaggctc cctgaggctg 60

agctgtgctg cctccggctt caccttcgac gactacggca tgtcctgggt gaggcaggct 120

cctggaaagg gcctggagtg ggtgtccggc atcaactgga acggcggatc caccggctac 180

gccgattccg tgaagggcag gttcaccatc agcagggaca acgccaagaa ctccctgtac 240

ctgcagatga actccctgag ggccgaggac accgccgtgt actactgcgc caggggcagg 300

tccctgctgt tcgactactg gggacagggc accctggtga ccgtgtccag g 351

<210> 219

<400> 219

000

<210> 220

<400> 220

000

<210> 221

<211> 508

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 221

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn

20 25 30

Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe

35 40 45

Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile

50 55 60

Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val

65 70 75 80

Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn

85 90 95

Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp

100 105 110

Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp

115 120 125

Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala

130 135 140

Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp

145 150 155 160

Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp Ser

165 170 175

Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr

180 185 190

Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val

195 200 205

Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys

210 215 220

Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys

225 230 235 240

Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly

245 250 255

Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser

260 265 270

Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile

275 280 285

Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp

290 295 300

Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp

305 310 315 320

Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser

325 330 335

Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile

340 345 350

Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile

355 360 365

Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys

370 375 380

Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Asn Trp Val Asn Val Ile

385 390 395 400

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

405 410 415

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

420 425 430

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser

435 440 445

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

450 455 460

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

465 470 475 480

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

485 490 495

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

500 505

<210> 222

<211> 18

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 222

Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly

<210> 223

<211> 823

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 223

Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr

1 5 10 15

Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu

20 25 30

Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly

35 40 45

Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly

50 55 60

Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu

65 70 75 80

Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys

85 90 95

Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys

100 105 110

Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr

115 120 125

Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln

130 135 140

Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly

145 150 155 160

Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala

165 170 175

Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala

180 185 190

Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg

195 200 205

Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro Leu

210 215 220

Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr Trp

225 230 235 240

Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val Gln

245 250 255

Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys Thr

260 265 270

Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg Ala

275 280 285

Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val Pro

290 295 300

Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

305 310 315 320

Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys

325 330 335

Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln

340 345 350

Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile

355 360 365

Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys

370 375 380

Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu

385 390 395 400

Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser

405 410 415

Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met

420 425 430

Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro

435 440 445

Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu

450 455 460

Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser

465 470 475 480

Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile

485 490 495

Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met

500 505 510

Ser Tyr Leu Asn Ala Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu

515 520 525

His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg

530 535 540

Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu

545 550 555 560

Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr

565 570 575

Pro Ser Leu Lys Cys Ile Arg Ser Glu Leu Thr Gln Asp Pro Ala Val

580 585 590

Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser

595 600 605

Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala

610 615 620

Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro

625 630 635 640

Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile

645 650 655

Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg

660 665 670

Asp Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys Leu Thr

675 680 685

Val Gly His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

690 695 700

Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro

705 710 715 720

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp

725 730 735

Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

740 745 750

Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp

755 760 765

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

770 775 780

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

785 790 795 800

Tyr Cys Ala Arg Gly Arg Ser Leu Leu Phe Asp Tyr Trp Gly Gln Gly

805 810 815

Thr Leu Val Thr Val Ser Arg

820

<210> 224

<211> 2469

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 224

atttgggaac tgaagaagga cgtctacgtg gtcgaactgg actggtatcc cgatgctccc 60

ggcgaaatgg tggtgctcac ttgtgacacc cccgaagaag acggcatcac ttggaccctc 120

gatcagagca gcgaggtgct gggctccgga aagaccctca caatccaagt taaggagttc 180

ggagacgctg gccaatacac atgccacaag ggaggcgagg tgctcagcca ttccttatta 240

ttattacaca agaaggaaga cggaatctgg tccaccgaca ttttaaaaga tcagaaggag 300

cccaagaata agaccttttt aaggtgtgag gccaaaaact acagcggtcg tttcacttgt 360

tggtggctga ccaccatttc caccgattta accttctccg tgaaaagcag ccggggaagc 420

tccgaccctc aaggtgtgac atgtggagcc gctaccctca gcgctgagag ggttcgtggc 480

gataacaagg aatacgagta cagcgtggag tgccaagaag atagcgcttg tcccgctgcc 540

gaagaatctt tacccattga ggtgatggtg gacgccgtgc acaaactcaa gtacgagaac 600

tacacctcct ccttctttat ccgggacatc attaagcccg atcctcctaa gaatttacag 660

ctgaagcctc tcaaaaatag ccggcaagtt gaggtctctt gggaatatcc cgacacttgg 720

agcacacccc acagctactt ctctttaacc ttttgtgtgc aagttcaagg taaaagcaag 780

cgggagaaga aagaccgggt gtttaccgac aaaaccagcg ccaccgtcat ctgtcggaag 840

aacgcctcca tcagcgtgag ggctcaagat cgttattact ccagcagctg gtccgagtgg 900

gccagcgtgc cttgttccgg cggtggagga tccggaggag gtggctccgg cggcggagga 960

tctcgtaacc tccccgtggc tacccccgat cccggaatgt tcccttgttt acaccacagc 1020

cagaatttac tgagggccgt gagcaacatg ctgcagaaag ctaggcagac tttagaattt 1080

tacccttgca ccagcgagga gatcgaccat gaagatatca ccaaggacaa gacatccacc 1140

gtggaggctt gtttacctct ggagctgaca aagaacgagt cttgtctcaa ctctcgtgaa 1200

accagcttca tcacaaatgg ctcttgttta gcttcccgga agacctcctt tatgatggct 1260

ttatgcctca gctccatcta cgaggattta aagatgtacc aagtggagtt caagaccatg 1320

aacgccaagc tgctcatgga ccctaaacgg cagatctttt tagaccagaa catgctggct 1380

gtgattgatg agctgatgca agctttaaac ttcaactccg agaccgtccc tcagaagtcc 1440

tccctcgagg agcccgattt ttacaagaca aagatcaaac tgtgcatttt actccacgcc 1500

tttaggatcc gggccgtgac cattgaccgg gtcatgagct atttaaacgc cagcattaca 1560

tgcccccctc ccatgagcgt ggagcacgcc gacatctggg tgaagagcta tagcctctac 1620

agccgggaga ggtatatctg taacagcggc ttcaagagga aggccggcac cagcagcctc 1680

accgagtgcg tgctgaataa ggctaccaac gtggctcact ggacaacacc ctctttaaag 1740

tgcatccggt ccgagctgac ccaggaccct gctgtgtccg tggctctggg ccagaccgtg 1800

aggatcacct gccagggcga ctccctgagg tcctactacg cctcctggta ccagcagaag 1860

cccggccagg ctcctgtgct ggtgatctac ggcaagaaca acaggccctc cggcatccct 1920

gacaggttct ccggatcctc ctccggcaac accgcctccc tgaccatcac aggcgctcag 1980

gccgaggacg aggctgacta ctactgcaac tccagggact cctccggcaa ccatgtggtg 2040

ttcggcggcg gcaccaagct gaccgtgggc catggcggcg gcggctccgg aggcggcggc 2100

agcggcggag gaggatccga ggtgcagctg gtggagtccg gaggaggagt ggtgaggcct 2160

ggaggctccc tgaggctgag ctgtgctgcc tccggcttca ccttcgacga ctacggcatg 2220

tcctgggtga ggcaggctcc tggaaagggc ctggagtggg tgtccggcat caactggaac 2280

ggcggatcca ccggctacgc cgattccgtg aagggcaggt tcaccatcag cagggacaac 2340

gccaagaact ccctgtacct gcagatgaac tccctgaggg ccgaggacac cgccgtgtac 2400

tactgcgcca ggggcaggtc cctgctgttc gactactggg gacagggcac cctggtgacc 2460

gtgtccagg 2469

<210> 225

<211> 841

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 225

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp

20 25 30

Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr

35 40 45

Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val

50 55 60

Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp

65 70 75 80

Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser

85 90 95

Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile

100 105 110

Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu

115 120 125

Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile

130 135 140

Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp

145 150 155 160

Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val

165 170 175

Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp

180 185 190

Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val

195 200 205

Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe

210 215 220

Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys

225 230 235 240

Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp

245 250 255

Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln

260 265 270

Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp

275 280 285

Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val

290 295 300

Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser

305 310 315 320

Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

325 330 335

Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe

340 345 350

Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met

355 360 365

Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu

370 375 380

Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu

385 390 395 400

Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser

405 410 415

Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys

420 425 430

Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu

435 440 445

Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met

450 455 460

Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile

465 470 475 480

Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln

485 490 495

Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu

500 505 510

Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg

515 520 525

Val Met Ser Tyr Leu Asn Ala Ser Ile Thr Cys Pro Pro Pro Met Ser

530 535 540

Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg

545 550 555 560

Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser

565 570 575

Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp

580 585 590

Thr Thr Pro Ser Leu Lys Cys Ile Arg Ser Glu Leu Thr Gln Asp Pro

595 600 605

Ala Val Ser Val Ala Leu Gly Gln Thr Val Arg Ile Thr Cys Gln Gly

610 615 620

Asp Ser Leu Arg Ser Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly

625 630 635 640

Gln Ala Pro Val Leu Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly

645 650 655

Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu

660 665 670

Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Asn

675 680 685

Ser Arg Asp Ser Ser Gly Asn His Val Val Phe Gly Gly Gly Thr Lys

690 695 700

Leu Thr Val Gly His Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

705 710 715 720

Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val

725 730 735

Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr

740 745 750

Phe Asp Asp Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly

755 760 765

Leu Glu Trp Val Ser Gly Ile Asn Trp Asn Gly Gly Ser Thr Gly Tyr

770 775 780

Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys

785 790 795 800

Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala

805 810 815

Val Tyr Tyr Cys Ala Arg Gly Arg Ser Leu Leu Phe Asp Tyr Trp Gly

820 825 830

Gln Gly Thr Leu Val Thr Val Ser Arg

835 840

<210> 226

<211> 2523

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 226

atgaaatggg tgacctttat ttctttactg ttcctcttta gcagcgccta ctccatttgg 60

gaactgaaga aggacgtcta cgtggtcgaa ctggactggt atcccgatgc tcccggcgaa 120

atggtggtgc tcacttgtga cacccccgaa gaagacggca tcacttggac cctcgatcag 180

agcagcgagg tgctgggctc cggaaagacc ctcacaatcc aagttaagga gttcggagac 240

gctggccaat acacatgcca caagggaggc gaggtgctca gccattcctt attattatta 300

cacaagaagg aagacggaat ctggtccacc gacattttaa aagatcagaa ggagcccaag 360

aataagacct ttttaaggtg tgaggccaaa aactacagcg gtcgtttcac ttgttggtgg 420

ctgaccacca tttccaccga tttaaccttc tccgtgaaaa gcagccgggg aagctccgac 480

cctcaaggtg tgacatgtgg agccgctacc ctcagcgctg agagggttcg tggcgataac 540

aaggaatacg agtacagcgt ggagtgccaa gaagatagcg cttgtcccgc tgccgaagaa 600

tctttaccca ttgaggtgat ggtggacgcc gtgcacaaac tcaagtacga gaactacacc 660

tcctccttct ttatccggga catcattaag cccgatcctc ctaagaattt acagctgaag 720

cctctcaaaa atagccggca agttgaggtc tcttgggaat atcccgacac ttggagcaca 780

ccccacagct acttctcttt aaccttttgt gtgcaagttc aaggtaaaag caagcgggag 840

aagaaagacc gggtgtttac cgacaaaacc agcgccaccg tcatctgtcg gaagaacgcc 900

tccatcagcg tgagggctca agatcgttat tactccagca gctggtccga gtgggccagc 960

gtgccttgtt ccggcggtgg aggatccgga ggaggtggct ccggcggcgg aggatctcgt 1020

aacctccccg tggctacccc cgatcccgga atgttccctt gtttacacca cagccagaat 1080

ttactgaggg ccgtgagcaa catgctgcag aaagctaggc agactttaga attttaccct 1140

tgcaccagcg aggagatcga ccatgaagat atcaccaagg acaagacatc caccgtggag 1200

gcttgtttac ctctggagct gacaaagaac gagtcttgtc tcaactctcg tgaaaccagc 1260

ttcatcacaa atggctcttg tttagcttcc cggaagacct cctttatgat ggctttatgc 1320

ctcagctcca tctacgagga tttaaagatg taccaagtgg agttcaagac catgaacgcc 1380

aagctgctca tggaccctaa acggcagatc tttttagacc agaacatgct ggctgtgatt 1440

gatgagctga tgcaagcttt aaacttcaac tccgagaccg tccctcagaa gtcctccctc 1500

gaggagcccg atttttacaa gacaaagatc aaactgtgca ttttactcca cgcctttagg 1560

atccgggccg tgaccattga ccgggtcatg agctatttaa acgccagcat tacatgcccc 1620

cctcccatga gcgtggagca cgccgacatc tgggtgaaga gctatagcct ctacagccgg 1680

gagaggtata tctgtaacag cggcttcaag aggaaggccg gcaccagcag cctcaccgag 1740

tgcgtgctga ataaggctac caacgtggct cactggacaa caccctcttt aaagtgcatc 1800

cggtccgagc tgacccagga ccctgctgtg tccgtggctc tgggccagac cgtgaggatc 1860

acctgccagg gcgactccct gaggtcctac tacgcctcct ggtaccagca gaagcccggc 1920

caggctcctg tgctggtgat ctacggcaag aacaacaggc cctccggcat ccctgacagg 1980

ttctccggat cctcctccgg caacaccgcc tccctgacca tcacaggcgc tcaggccgag 2040

gacgaggctg actactactg caactccagg gactcctccg gcaaccatgt ggtgttcggc 2100

ggcggcacca agctgaccgt gggccatggc ggcggcggct ccggaggcgg cggcagcggc 2160

ggaggaggat ccgaggtgca gctggtggag tccggaggag gagtggtgag gcctggaggc 2220

tccctgaggc tgagctgtgc tgcctccggc ttcaccttcg acgactacgg catgtcctgg 2280

gtgaggcagg ctcctggaaa gggcctggag tgggtgtccg gcatcaactg gaacggcgga 2340

tccaccggct acgccgattc cgtgaagggc aggttcacca tcagcaggga caacgccaag 2400

aactccctgt acctgcagat gaactccctg agggccgagg acaccgccgt gtactactgc 2460

gccaggggca ggtccctgct gttcgactac tggggacagg gcaccctggt gaccgtgtcc 2520

agg 2523

<210> 227

<211> 456

<212> DNA

<213> Chile person

<400> 227

gattgcgaca tcgagggcaa ggacggcaag cagtacgaga gcgtgctgat ggtgtccatc 60

gaccagctgc tggacagcat gaaggagatc ggctccaact gcctcaacaa cgagttcaac 120

ttcttcaagc ggcacatctg cgacgccaac aaggagggca tgttcctgtt cagggccgcc 180

aggaaactgc ggcagttcct gaagatgaac tccaccggcg acttcgacct gcacctgctg 240

aaggtgtccg agggcaccac catcctgctg aactgcaccg gacaggtgaa gggccggaaa 300

cctgctgctc tgggagaggc ccaacccacc aagagcctgg aggagaacaa gtccctgaag 360

gagcagaaga agctgaacga cctgtgcttc ctgaagaggc tgctgcagga gatcaagacc 420

tgctggaaca agatcctgat gggcaccaag gagcat 456

<210> 228

<400> 228

000

<210> 229

<400> 229

000

<210> 230

<400> 230

000

<210> 231

<400> 231

000

<210> 232

<211> 438

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 232

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr

1 5 10 15

Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly

35 40 45

Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser

50 55 60

Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val

85 90 95

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Gly His Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met Ser Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Trp

165 170 175

Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Ser

210 215 220

Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Arg

225 230 235 240

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

245 250 255

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

260 265 270

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

275 280 285

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

290 295 300

Arg Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp

305 310 315 320

Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe

325 330 335

Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe

340 345 350

Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn

355 360 365

Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro

370 375 380

Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser

385 390 395 400

Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe

405 410 415

Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr

420 425 430

His Gly Ser Glu Asp Ser

435

<210> 233

<211> 1314

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 233

tccgagctga cccaggaccc tgctgtgtcc gtggctctgg gccagaccgt gaggatcacc 60

tgccagggcg actccctgag gtcctactac gcctcctggt accagcagaa gcccggccag 120

gctcctgtgc tggtgatcta cggcaagaac aacaggccct ccggcatccc tgacaggttc 180

tccggatcct cctccggcaa caccgcctcc ctgaccatca caggcgctca ggccgaggac 240

gaggctgact actactgcaa ctccagggac tcctccggca accatgtggt gttcggcggc 300

ggcaccaagc tgaccgtggg ccatggcggc ggcggctccg gaggcggcgg cagcggcgga 360

ggaggatccg aggtgcagct ggtggagtcc ggaggaggag tggtgaggcc tggaggctcc 420

ctgaggctga gctgtgctgc ctccggcttc accttcgacg actacggcat gtcctgggtg 480

aggcaggctc ctggaaaggg cctggagtgg gtgtccggca tcaactggaa cggcggatcc 540

accggctacg ccgattccgt gaagggcagg ttcaccatca gcagggacaa cgccaagaac 600

tccctgtacc tgcagatgaa ctccctgagg gccgaggaca ccgccgtgta ctactgcgcc 660

aggggcaggt ccctgctgtt cgactactgg ggacagggca ccctggtgac cgtgtccagg 720

attacatgcc cccctcccat gagcgtggag cacgccgaca tctgggtgaa gagctatagc 780

ctctacagcc gggagaggta tatctgtaac agcggcttca agaggaaggc cggcaccagc 840

agcctcaccg agtgcgtgct gaataaggct accaacgtgg ctcactggac aacaccctct 900

ttaaagtgca tccggcaggg ccaggacagg cacatgatcc ggatgaggca gctcatcgac 960

atcgtcgacc agctgaagaa ctacgtgaac gacctggtgc ccgagtttct gcctgccccc 1020

gaggacgtgg agaccaactg cgagtggtcc gccttctcct gctttcagaa ggcccagctg 1080

aagtccgcca acaccggcaa caacgagcgg atcatcaacg tgagcatcaa gaagctgaag 1140

cggaagcctc cctccacaaa cgccggcagg aggcagaagc acaggctgac ctgccccagc 1200

tgtgactcct acgagaagaa gccccccaag gagttcctgg agaggttcaa gtccctgctg 1260

cagaagatga tccatcagca cctgtcctcc aggacccacg gctccgagga ctcc 1314

<210> 234

<211> 456

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 234

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly

20 25 30

Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr

35 40 45

Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile

50 55 60

Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly

65 70 75 80

Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala

85 90 95

Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn

100 105 110

His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Gly His Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

130 135 140

Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met Ser

165 170 175

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile

180 185 190

Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly Arg

195 200 205

Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met

210 215 220

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly

225 230 235 240

Arg Ser Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

245 250 255

Ser Arg Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

260 265 270

Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

275 280 285

Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val

290 295 300

Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

305 310 315 320

Cys Ile Arg Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu

325 330 335

Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro

340 345 350

Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser

355 360 365

Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly

370 375 380

Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys

385 390 395 400

Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys

405 410 415

Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu

420 425 430

Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser

435 440 445

Arg Thr His Gly Ser Glu Asp Ser

450 455

<210> 235

<211> 1368

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 235

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctcctccgag 60

ctgacccagg accctgctgt gtccgtggct ctgggccaga ccgtgaggat cacctgccag 120

ggcgactccc tgaggtccta ctacgcctcc tggtaccagc agaagcccgg ccaggctcct 180

gtgctggtga tctacggcaa gaacaacagg ccctccggca tccctgacag gttctccgga 240

tcctcctccg gcaacaccgc ctccctgacc atcacaggcg ctcaggccga ggacgaggct 300

gactactact gcaactccag ggactcctcc ggcaaccatg tggtgttcgg cggcggcacc 360

aagctgaccg tgggccatgg cggcggcggc tccggaggcg gcggcagcgg cggaggagga 420

tccgaggtgc agctggtgga gtccggagga ggagtggtga ggcctggagg ctccctgagg 480

ctgagctgtg ctgcctccgg cttcaccttc gacgactacg gcatgtcctg ggtgaggcag 540

gctcctggaa agggcctgga gtgggtgtcc ggcatcaact ggaacggcgg atccaccggc 600

tacgccgatt ccgtgaaggg caggttcacc atcagcaggg acaacgccaa gaactccctg 660

tacctgcaga tgaactccct gagggccgag gacaccgccg tgtactactg cgccaggggc 720

aggtccctgc tgttcgacta ctggggacag ggcaccctgg tgaccgtgtc caggattaca 780

tgcccccctc ccatgagcgt ggagcacgcc gacatctggg tgaagagcta tagcctctac 840

agccgggaga ggtatatctg taacagcggc ttcaagagga aggccggcac cagcagcctc 900

accgagtgcg tgctgaataa ggctaccaac gtggctcact ggacaacacc ctctttaaag 960

tgcatccggc agggccagga caggcacatg atccggatga ggcagctcat cgacatcgtc 1020

gaccagctga agaactacgt gaacgacctg gtgcccgagt ttctgcctgc ccccgaggac 1080

gtggagacca actgcgagtg gtccgccttc tcctgctttc agaaggccca gctgaagtcc 1140

gccaacaccg gcaacaacga gcggatcatc aacgtgagca tcaagaagct gaagcggaag 1200

cctccctcca caaacgccgg caggaggcag aagcacaggc tgacctgccc cagctgtgac 1260

tcctacgaga agaagccccc caaggagttc ctggagaggt tcaagtccct gctgcagaag 1320

atgatccatc agcacctgtc ctccaggacc cacggctccg aggactcc 1368

<210> 236

<211> 620

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 236

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val

145 150 155 160

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

165 170 175

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

180 185 190

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

195 200 205

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

210 215 220

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

225 230 235 240

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

245 250 255

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

260 265 270

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ser

275 280 285

Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr

290 295 300

Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val

305 310 315 320

Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys

325 330 335

Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys

340 345 350

Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly

355 360 365

Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser

370 375 380

Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile

385 390 395 400

Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp

405 410 415

Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp

420 425 430

Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser

435 440 445

Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile

450 455 460

Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile

465 470 475 480

Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys

485 490 495

Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Asn Trp Val Asn Val Ile

500 505 510

Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp

515 520 525

Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr

530 535 540

Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser

545 550 555 560

Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala

565 570 575

Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys

580 585 590

Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser

595 600 605

Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

610 615 620

<210> 237

<211> 1860

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 237

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcagcatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgctcc 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga cagcggcaca accaacacag tcgctgccta taacctcact 900

tggaagagca ccaacttcaa aaccatcctc gaatgggaac ccaaacccgt taaccaagtt 960

tacaccgtgc agatcagcac caagtccggc gactggaagt ccaaatgttt ctataccacc 1020

gacaccgagt gcgatctcac cgatgagatc gtgaaagatg tgaaacagac ctacctcgcc 1080

cgggtgttta gctaccccgc cggcaatgtg gagagcactg gttccgctgg cgagccttta 1140

tacgagaaca gccccgaatt taccccttac ctcgagacca atttaggaca gcccaccatc 1200

caaagctttg agcaagttgg cacaaaggtg aatgtgacag tggaggacga gcggacttta 1260

gtgcggcgga acaacacctt tctcagcctc cgggatgtgt tcggcaaaga tttaatctac 1320

acactgtatt actggaagtc ctcttcctcc ggcaagaaga cagctaaaac caacacaaac 1380

gagtttttaa tcgacgtgga taaaggcgaa aactactgtt tcagcgtgca agctgtgatc 1440

ccctcccgga ccgtgaatag gaaaagcacc gatagccccg ttgagtgcat gggccaagaa 1500

aagggcgagt tccgggagaa ctgggtgaac gtcatcagcg atttaaagaa gatcgaagat 1560

ttaattcagt ccatgcatat cgacgccact ttatacacag aatccgacgt gcacccctct 1620

tgtaaggtga ccgccatgaa atgtttttta ctggagctgc aagttatctc tttagagagc 1680

ggagacgcta gcatccacga caccgtggag aatttaatca ttttagccaa taactcttta 1740

tccagcaacg gcaacgtgac agagtccggc tgcaaggagt gcgaagagct ggaggagaag 1800

aacatcaagg agtttctgca atcctttgtg cacattgtcc agatgttcat caatacctcc 1860

<210> 238

<211> 638

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 238

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

20 25 30

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

35 40 45

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

50 55 60

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

65 70 75 80

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

85 90 95

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

100 105 110

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

115 120 125

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

130 135 140

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys

165 170 175

Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys

180 185 190

Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp

195 200 205

Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu

210 215 220

Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn

225 230 235 240

Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp

245 250 255

Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys

260 265 270

Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu

275 280 285

Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro

290 295 300

Asp Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys

305 310 315 320

Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn

325 330 335

Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser

340 345 350

Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile

355 360 365

Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro

370 375 380

Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu

385 390 395 400

Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro

405 410 415

Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val

420 425 430

Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu

435 440 445

Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys

450 455 460

Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe

465 470 475 480

Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala

485 490 495

Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val

500 505 510

Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Asn Trp Val Asn

515 520 525

Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His

530 535 540

Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys

545 550 555 560

Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu

565 570 575

Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile

580 585 590

Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly

595 600 605

Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu

610 615 620

Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr Ser

625 630 635

<210> 239

<211> 1914

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 239

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccatcccc 60

ccccatgtgc aaaagagcgt gaacaacgat atgatcgtga ccgacaacaa cggcgccgtg 120

aagtttcccc agctctgcaa gttctgcgat gtcaggttca gcacctgcga taatcagaag 180

tcctgcatgt ccaactgcag catcacctcc atctgcgaga agccccaaga agtgtgcgtg 240

gccgtgtggc ggaaaaatga cgagaacatc accctggaga ccgtgtgtca cgaccccaag 300

ctcccttatc acgacttcat tctggaggac gctgcctccc ccaaatgcat catgaaggag 360

aagaagaagc ccggagagac cttctttatg tgttcctgta gcagcgacga gtgtaacgac 420

aacatcatct tcagcgaaga gtacaacacc agcaaccctg atggaggtgg cggatccgga 480

ggtggaggtt ctggtggagg tgggagtatt cctccccacg tgcagaagag cgtgaataat 540

gacatgatcg tgaccgataa caatggcgcc gtgaaatttc cccagctgtg caaattctgc 600

gatgtgaggt tttccacctg cgacaaccag aagtcctgta tgagcaactg ctccatcacc 660

tccatctgtg agaagcctca ggaggtgtgc gtggctgtct ggcggaagaa tgacgagaat 720

atcaccctgg aaaccgtctg ccacgatccc aagctgccct accacgattt catcctggaa 780

gacgccgcca gccctaagtg catcatgaaa gagaaaaaga agcctggcga gacctttttc 840

atgtgctcct gcagcagcga cgaatgcaac gacaatatca tctttagcga ggaatacaat 900

accagcaacc ccgacagcgg cacaaccaac acagtcgctg cctataacct cacttggaag 960

agcaccaact tcaaaaccat cctcgaatgg gaacccaaac ccgttaacca agtttacacc 1020

gtgcagatca gcaccaagtc cggcgactgg aagtccaaat gtttctatac caccgacacc 1080

gagtgcgatc tcaccgatga gatcgtgaaa gatgtgaaac agacctacct cgcccgggtg 1140

tttagctacc ccgccggcaa tgtggagagc actggttccg ctggcgagcc tttatacgag 1200

aacagccccg aatttacccc ttacctcgag accaatttag gacagcccac catccaaagc 1260

tttgagcaag ttggcacaaa ggtgaatgtg acagtggagg acgagcggac tttagtgcgg 1320

cggaacaaca cctttctcag cctccgggat gtgttcggca aagatttaat ctacacactg 1380

tattactgga agtcctcttc ctccggcaag aagacagcta aaaccaacac aaacgagttt 1440

ttaatcgacg tggataaagg cgaaaactac tgtttcagcg tgcaagctgt gatcccctcc 1500

cggaccgtga ataggaaaag caccgatagc cccgttgagt gcatgggcca agaaaagggc 1560

gagttccggg agaactgggt gaacgtcatc agcgatttaa agaagatcga agatttaatt 1620

cagtccatgc atatcgacgc cactttatac acagaatccg acgtgcaccc ctcttgtaag 1680

gtgaccgcca tgaaatgttt tttactggag ctgcaagtta tctctttaga gagcggagac 1740

gctagcatcc acgacaccgt ggagaattta atcattttag ccaataactc tttatccagc 1800

aacggcaacg tgacagagtc cggctgcaag gagtgcgaag agctggagga gaagaacatc 1860

aaggagtttc tgcaatcctt tgtgcacatt gtccagatgt tcatcaatac ctcc 1914

<210> 240

<211> 352

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 240

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val

145 150 155 160

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

165 170 175

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

180 185 190

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

195 200 205

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

210 215 220

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

225 230 235 240

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

245 250 255

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

260 265 270

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile

275 280 285

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

290 295 300

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

305 310 315 320

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

325 330 335

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

340 345 350

<210> 241

<211> 1056

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 241

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcagcatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgctcc 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga cattacatgc ccccctccca tgagcgtgga gcacgccgac 900

atctgggtga agagctatag cctctacagc cgggagaggt atatctgtaa cagcggcttc 960

aagaggaagg ccggcaccag cagcctcacc gagtgcgtgc tgaataaggc taccaacgtg 1020

gctcactgga caacaccctc tttaaagtgc atccgg 1056

<210> 242

<211> 370

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 242

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

20 25 30

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

35 40 45

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

50 55 60

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

65 70 75 80

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

85 90 95

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

100 105 110

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

115 120 125

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

130 135 140

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys

165 170 175

Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys

180 185 190

Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp

195 200 205

Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu

210 215 220

Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn

225 230 235 240

Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp

245 250 255

Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys

260 265 270

Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu

275 280 285

Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro

290 295 300

Asp Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

305 310 315 320

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

325 330 335

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

340 345 350

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

355 360 365

Ile Arg

370

<210> 243

<211> 1110

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 243

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccatcccc 60

ccccatgtgc aaaagagcgt gaacaacgat atgatcgtga ccgacaacaa cggcgccgtg 120

aagtttcccc agctctgcaa gttctgcgat gtcaggttca gcacctgcga taatcagaag 180

tcctgcatgt ccaactgcag catcacctcc atctgcgaga agccccaaga agtgtgcgtg 240

gccgtgtggc ggaaaaatga cgagaacatc accctggaga ccgtgtgtca cgaccccaag 300

ctcccttatc acgacttcat tctggaggac gctgcctccc ccaaatgcat catgaaggag 360

aagaagaagc ccggagagac cttctttatg tgttcctgta gcagcgacga gtgtaacgac 420

aacatcatct tcagcgaaga gtacaacacc agcaaccctg atggaggtgg cggatccgga 480

ggtggaggtt ctggtggagg tgggagtatt cctccccacg tgcagaagag cgtgaataat 540

gacatgatcg tgaccgataa caatggcgcc gtgaaatttc cccagctgtg caaattctgc 600

gatgtgaggt tttccacctg cgacaaccag aagtcctgta tgagcaactg ctccatcacc 660

tccatctgtg agaagcctca ggaggtgtgc gtggctgtct ggcggaagaa tgacgagaat 720

atcaccctgg aaaccgtctg ccacgatccc aagctgccct accacgattt catcctggaa 780

gacgccgcca gccctaagtg catcatgaaa gagaaaaaga agcctggcga gacctttttc 840

atgtgctcct gcagcagcga cgaatgcaac gacaatatca tctttagcga ggaatacaat 900

accagcaacc ccgacattac atgcccccct cccatgagcg tggagcacgc cgacatctgg 960

gtgaagagct atagcctcta cagccgggag aggtatatct gtaacagcgg cttcaagagg 1020

aaggccggca ccagcagcct caccgagtgc gtgctgaata aggctaccaa cgtggctcac 1080

tggacaacac cctctttaaa gtgcatccgg 1110

<210> 244

<211> 1911

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 244

atgggagtga aagttctttt tgcccttatt tgtattgctg tggccgaggc catcccaccg 60

cacgttcaga agtcggtgaa taacgacatg atagtcactg acaacaacgg tgcagtcaag 120

tttccacaac tgtgtaaatt ttgtgatgtg agattttcca cctgtgacaa ccagaaatcc 180

tgcatgagca actgcagcat cacctccatc tgtgagaagc cacaggaagt ctgtgtggct 240

gtatggagaa agaatgacga gaacataaca ctagagacag tttgccatga ccccaagctc 300

ccctaccatg actttattct ggaagatgct gcttctccaa agtgcattat gaaggaaaaa 360

aaaaagcctg gtgagacttt cttcatgtgt tcctgtagct ctgatgagtg caatgacaac 420

atcatcttct cagaagaata taacaccagc aatcctgacg gaggtggcgg atccggaggt 480

ggaggttctg gtggaggtgg gagtattcct ccccacgtgc agaagagcgt gaataatgac 540

atgatcgtga ccgataacaa tggcgccgtg aaatttcccc agctgtgcaa attctgcgat 600

gtgaggtttt ccacctgcga caaccagaag tcctgtatga gcaactgctc catcacctcc 660

atctgtgaga agcctcagga ggtgtgcgtg gctgtctggc ggaagaatga cgagaatatc 720

accctggaaa ccgtctgcca cgatcccaag ctgccctacc acgatttcat cctggaagac 780

gccgccagcc ctaagtgcat catgaaagag aaaaagaagc ctggcgagac ctttttcatg 840

tgctcctgca gcagcgacga atgcaacgac aatatcatct ttagcgagga atacaatacc 900

agcaaccccg actcaggcac tacaaatact gtggcagcat ataatttaac ttggaaatca 960

actaatttca agacaatttt ggagtgggaa cccaaacccg tcaatcaagt ctacactgtt 1020

caaataagca ctaagtcagg agattggaaa agcaaatgct tttacacaac agacacagag 1080

tgtgacctca ccgacgagat tgtgaaggat gtgaagcaga cgtacttggc acgggtcttc 1140

tcctacccgg cagggaatgt ggagagcacc ggttctgctg gggagcctct gtatgagaac 1200

tccccagagt tcacacctta cctggagaca aacctcggac agccaacaat tcagagtttt 1260

gaacaggtgg gaacaaaagt gaatgtgacc gtagaagatg aacggacttt agtcagaagg 1320

aacaacactt tcctaagcct ccgggatgtt tttggcaagg acttaattta tacactttat 1380

tattggaaat cttcaagttc aggaaagaaa acagccaaaa caaacactaa tgagtttttg 1440

attgatgtgg ataaaggaga aaactactgt ttcagtgttc aagcagtgat tccctcccga 1500

acagttaacc ggaagagtac agacagcccg gtagagtgta tgggccagga gaaaggggaa 1560

ttcagagaaa actgggtgaa tgtaataagt aatttgaaaa aaattgaaga tcttattcaa 1620

tctatgcata ttgatgctac tttatatacg gaaagtgatg ttcaccccag ttgcaaagta 1680

acagcaatga agtgctttct cttggagtta caagttattt cacttgagtc cggagatgca 1740

agtattcatg atacagtaga aaatctgatc atcctagcaa acaacagttt gtcttctaat 1800

gggaatgtaa cagaatctgg atgcaaagaa tgtgaggaac tggaggaaaa aaatattaaa 1860

gaatttttgc agagttttgt acatattgtc caaatgttca tcaacacttc t 1911

<210> 245

<400> 245

000

<210> 246

<400> 246

000

<210> 247

<400> 247

000

<210> 248

<400> 248

000

<210> 249

<400> 249

000

<210> 250

<211> 235

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 250

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser

20 25 30

Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile

35 40 45

Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile

50 55 60

Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys

65 70 75 80

Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His

85 90 95

Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly

100 105 110

Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr

115 120 125

Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn

130 135 140

Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp

145 150 155 160

Asn Lys Ile Leu Met Gly Thr Lys Glu His Ile Thr Cys Pro Pro Pro

165 170 175

Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr

180 185 190

Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly

195 200 205

Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala

210 215 220

His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

225 230 235

<210> 251

<211> 705

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 251

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccgattgc 60

gacatcgagg gcaaggacgg caagcagtac gagagcgtgc tgatggtgtc catcgaccag 120

ctgctggaca gcatgaagga gatcggctcc aactgcctca acaacgagtt caacttcttc 180

aagcggcaca tctgcgacgc caacaaggag ggcatgttcc tgttcagggc cgccaggaaa 240

ctgcggcagt tcctgaagat gaactccacc ggcgacttcg acctgcacct gctgaaggtg 300

tccgagggca ccaccatcct gctgaactgc accggacagg tgaagggccg gaaacctgct 360

gctctgggag aggcccaacc caccaagagc ctggaggaga acaagtccct gaaggagcag 420

aagaagctga acgacctgtg cttcctgaag aggctgctgc aggagatcaa gacctgctgg 480

aacaagatcc tgatgggcac caaggagcat attacatgcc cccctcccat gagcgtggag 540

cacgccgaca tctgggtgaa gagctatagc ctctacagcc gggagaggta tatctgtaac 600

agcggcttca agaggaaggc cggcaccagc agcctcaccg agtgcgtgct gaataaggct 660

accaacgtgg ctcactggac aacaccctct ttaaagtgca tccgg 705

<210> 252

<400> 252

000

<210> 253

<400> 253

000

<210> 254

<400> 254

000

<210> 255

<400> 255

000

<210> 256

<400> 256

000

<210> 257

<211> 1056

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 257

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcagcatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgctcc 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga cattacatgc ccccctccca tgagcgtgga gcacgccgac 900

atctgggtga agagctatag cctctacagc cgggagaggt atatctgtaa cagcggcttc 960

aagaggaagg ccggcaccag cagcctcacc gagtgcgtgc tgaataaggc taccaacgtg 1020

gctcactgga caacaccctc tttaaagtgc atccgg 1056

<210> 258

<400> 258

000

<210> 259

<211> 1110

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 259

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccatcccc 60

ccccatgtgc aaaagagcgt gaacaacgat atgatcgtga ccgacaacaa cggcgccgtg 120

aagtttcccc agctctgcaa gttctgcgat gtcaggttca gcacctgcga taatcagaag 180

tcctgcatgt ccaactgcag catcacctcc atctgcgaga agccccaaga agtgtgcgtg 240

gccgtgtggc ggaaaaatga cgagaacatc accctggaga ccgtgtgtca cgaccccaag 300

ctcccttatc acgacttcat tctggaggac gctgcctccc ccaaatgcat catgaaggag 360

aagaagaagc ccggagagac cttctttatg tgttcctgta gcagcgacga gtgtaacgac 420

aacatcatct tcagcgaaga gtacaacacc agcaaccctg atggaggtgg cggatccgga 480

ggtggaggtt ctggtggagg tgggagtatt cctccccacg tgcagaagag cgtgaataat 540

gacatgatcg tgaccgataa caatggcgcc gtgaaatttc cccagctgtg caaattctgc 600

gatgtgaggt tttccacctg cgacaaccag aagtcctgta tgagcaactg ctccatcacc 660

tccatctgtg agaagcctca ggaggtgtgc gtggctgtct ggcggaagaa tgacgagaat 720

atcaccctgg aaaccgtctg ccacgatccc aagctgccct accacgattt catcctggaa 780

gacgccgcca gccctaagtg catcatgaaa gagaaaaaga agcctggcga gacctttttc 840

atgtgctcct gcagcagcga cgaatgcaac gacaatatca tctttagcga ggaatacaat 900

accagcaacc ccgacattac atgcccccct cccatgagcg tggagcacgc cgacatctgg 960

gtgaagagct atagcctcta cagccgggag aggtatatct gtaacagcgg cttcaagagg 1020

aaggccggca ccagcagcct caccgagtgc gtgctgaata aggctaccaa cgtggctcac 1080

tggacaacac cctctttaaa gtgcatccgg 1110

<210> 260

<211> 184

<212> PRT

<213> Chile person

<400> 260

Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp

1 5 10 15

Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu

20 25 30

Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val

35 40 45

Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val

50 55 60

Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg

65 70 75 80

Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His

85 90 95

Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr

100 105 110

Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly

115 120 125

Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val

130 135 140

His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln

145 150 155 160

Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175

Gly Leu Pro Ser Pro Arg Ser Glu

180

<210> 261

<211> 552

<212> DNA

<213> Chile person

<400> 261

cgcgagggtc ccgagctttc gcccgacgat cccgccggcc tcttggacct gcggcagggc 60

atgtttgcgc agctggtggc ccaaaatgtt ctgctgatcg atgggcccct gagctggtac 120

agtgacccag gcctggcagg cgtgtccctg acggggggcc tgagctacaa agaggacacg 180

aaggagctgg tggtggccaa ggctggagtc tactatgtct tctttcaact agagctgcgg 240

cgcgtggtgg ccggcgaggg ctcaggctcc gtttcacttg cgctgcacct gcagccactg 300

cgctctgctg ctggggccgc cgccctggct ttgaccgtgg acctgccacc cgcctcctcc 360

gaggctcgga actcggcctt cggtttccag ggccgcttgc tgcacctgag tgccggccag 420

cgcctgggcg tccatcttca cactgaggcc agggcacgcc atgcctggca gcttacccag 480

ggcgccacag tcttgggact cttccgggtg acccccgaaa tcccagccgg actcccttca 540

ccgaggtcgg aa 552

<210> 262

<211> 165

<212> PRT

<213> Chile person

<400> 262

Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu

1 5 10 15

Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser

20 25 30

Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys

35 40 45

Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val

50 55 60

Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly

65 70 75 80

Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly

85 90 95

Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu

100 105 110

Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser

115 120 125

Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg

130 135 140

His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg

145 150 155 160

Val Thr Pro Glu Ile

165

<210> 263

<211> 495

<212> DNA

<213> Chile person

<400> 263

gatcccgccg gcctcttgga cctgcggcag ggcatgtttg cgcagctggt ggcccaaaat 60

gttctgctga tcgatgggcc cctgagctgg tacagtgacc caggcctggc aggcgtgtcc 120

ctgacggggg gcctgagcta caaagaggac acgaaggagc tggtggtggc caaggctgga 180

gtctactatg tcttctttca actagagctg cggcgcgtgg tggccggcga gggctcaggc 240

tccgtttcac ttgcgctgca cctgcagcca ctgcgctctg ctgctggggc cgccgccctg 300

gctttgaccg tggacctgcc acccgcctcc tccgaggctc ggaactcggc cttcggtttc 360

cagggccgct tgctgcacct gagtgccggc cagcgcctgg gcgtccatct tcacactgag 420

gccagggcac gccatgcctg gcagcttacc cagggcgcca cagtcttggg actcttccgg 480

gtgacccccg aaatc 495

<210> 264

<400> 264

000

<210> 265

<400> 265

000

<210> 266

<400> 266

000

<210> 267

<400> 267

000

<210> 268

<211> 397

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 268

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His

130 135 140

Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr

145 150 155 160

Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr

165 170 175

Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro

180 185 190

Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

195 200 205

Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro

210 215 220

Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala

225 230 235 240

Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro

245 250 255

Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp

260 265 270

Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe

275 280 285

Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val

290 295 300

Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala

305 310 315 320

Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg

325 330 335

Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly

340 345 350

Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala

355 360 365

Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr

370 375 380

Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

385 390 395

<210> 269

<211> 1191

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 269

cagggccagg acaggcacat gatccggatg aggcagctca tcgacatcgt cgaccagctg 60

aagaactacg tgaacgacct ggtgcccgag tttctgcctg cccccgagga cgtggagacc 120

aactgcgagt ggtccgcctt ctcctgcttt cagaaggccc agctgaagtc cgccaacacc 180

ggcaacaacg agcggatcat caacgtgagc atcaagaagc tgaagcggaa gcctccctcc 240

acaaacgccg gcaggaggca gaagcacagg ctgacctgcc ccagctgtga ctcctacgag 300

aagaagcccc ccaaggagtt cctggagagg ttcaagtccc tgctgcagaa gatgatccat 360

cagcacctgt cctccaggac ccacggctcc gaggactcca ttacatgccc ccctcccatg 420

agcgtggagc acgccgacat ctgggtgaag agctatagcc tctacagccg ggagaggtat 480

atctgtaaca gcggcttcaa gaggaaggcc ggcaccagca gcctcaccga gtgcgtgctg 540

aataaggcta ccaacgtggc tcactggaca acaccctctt taaagtgcat ccggggcggt 600

ggaggatccg gaggaggtgg ctccggcggc ggaggatctc gcgagggtcc cgagctttcg 660

cccgacgatc ccgccggcct cttggacctg cggcagggca tgtttgcgca gctggtggcc 720

caaaatgttc tgctgatcga tgggcccctg agctggtaca gtgacccagg cctggcaggc 780

gtgtccctga cggggggcct gagctacaaa gaggacacga aggagctggt ggtggccaag 840

gctggagtct actatgtctt ctttcaacta gagctgcggc gcgtggtggc cggcgagggc 900

tcaggctccg tttcacttgc gctgcacctg cagccactgc gctctgctgc tggggccgcc 960

gccctggctt tgaccgtgga cctgccaccc gcctcctccg aggctcggaa ctcggccttc 1020

ggtttccagg gccgcttgct gcacctgagt gccggccagc gcctgggcgt ccatcttcac 1080

actgaggcca gggcacgcca tgcctggcag cttacccagg gcgccacagt cttgggactc 1140

ttccgggtga cccccgaaat cccagccgga ctcccttcac cgaggtcgga a 1191

<210> 270

<211> 415

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 270

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

20 25 30

Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

35 40 45

Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

50 55 60

Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn

65 70 75 80

Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

85 90 95

Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

100 105 110

Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

115 120 125

Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg

130 135 140

Thr His Gly Ser Glu Asp Ser Ile Thr Cys Pro Pro Pro Met Ser Val

145 150 155 160

Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu

165 170 175

Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser

180 185 190

Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr

195 200 205

Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp

225 230 235 240

Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu

245 250 255

Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser

260 265 270

Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys

275 280 285

Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val

290 295 300

Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly

305 310 315 320

Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly

325 330 335

Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu

340 345 350

Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser

355 360 365

Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg

370 375 380

His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg

385 390 395 400

Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

405 410 415

<210> 271

<211> 1245

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 271

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctcccagggc 60

caggacaggc acatgatccg gatgaggcag ctcatcgaca tcgtcgacca gctgaagaac 120

tacgtgaacg acctggtgcc cgagtttctg cctgcccccg aggacgtgga gaccaactgc 180

gagtggtccg ccttctcctg ctttcagaag gcccagctga agtccgccaa caccggcaac 240

aacgagcgga tcatcaacgt gagcatcaag aagctgaagc ggaagcctcc ctccacaaac 300

gccggcagga ggcagaagca caggctgacc tgccccagct gtgactccta cgagaagaag 360

ccccccaagg agttcctgga gaggttcaag tccctgctgc agaagatgat ccatcagcac 420

ctgtcctcca ggacccacgg ctccgaggac tccattacat gcccccctcc catgagcgtg 480

gagcacgccg acatctgggt gaagagctat agcctctaca gccgggagag gtatatctgt 540

aacagcggct tcaagaggaa ggccggcacc agcagcctca ccgagtgcgt gctgaataag 600

gctaccaacg tggctcactg gacaacaccc tctttaaagt gcatccgggg cggtggagga 660

tccggaggag gtggctccgg cggcggagga tctcgcgagg gtcccgagct ttcgcccgac 720

gatcccgccg gcctcttgga cctgcggcag ggcatgtttg cgcagctggt ggcccaaaat 780

gttctgctga tcgatgggcc cctgagctgg tacagtgacc caggcctggc aggcgtgtcc 840

ctgacggggg gcctgagcta caaagaggac acgaaggagc tggtggtggc caaggctgga 900

gtctactatg tcttctttca actagagctg cggcgcgtgg tggccggcga gggctcaggc 960

tccgtttcac ttgcgctgca cctgcagcca ctgcgctctg ctgctggggc cgccgccctg 1020

gctttgaccg tggacctgcc acccgcctcc tccgaggctc ggaactcggc cttcggtttc 1080

cagggccgct tgctgcacct gagtgccggc cagcgcctgg gcgtccatct tcacactgag 1140

gccagggcac gccatgcctg gcagcttacc cagggcgcca cagtcttggg actcttccgg 1200

gtgacccccg aaatcccagc cggactccct tcaccgaggt cggaa 1245

<210> 272

<211> 378

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 272

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

1 5 10 15

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

20 25 30

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

35 40 45

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

50 55 60

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

65 70 75 80

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

85 90 95

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

100 105 110

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

115 120 125

Gly Ser Glu Asp Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His

130 135 140

Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr

145 150 155 160

Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr

165 170 175

Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro

180 185 190

Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

195 200 205

Gly Gly Gly Gly Ser Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly

210 215 220

Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro

225 230 235 240

Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly

245 250 255

Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala

260 265 270

Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala

275 280 285

Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu

290 295 300

Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro

305 310 315 320

Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg

325 330 335

Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr

340 345 350

Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val

355 360 365

Leu Gly Leu Phe Arg Val Thr Pro Glu Ile

370 375

<210> 273

<211> 1134

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 273

cagggccagg acaggcacat gatccggatg aggcagctca tcgacatcgt cgaccagctg 60

aagaactacg tgaacgacct ggtgcccgag tttctgcctg cccccgagga cgtggagacc 120

aactgcgagt ggtccgcctt ctcctgcttt cagaaggccc agctgaagtc cgccaacacc 180

ggcaacaacg agcggatcat caacgtgagc atcaagaagc tgaagcggaa gcctccctcc 240

acaaacgccg gcaggaggca gaagcacagg ctgacctgcc ccagctgtga ctcctacgag 300

aagaagcccc ccaaggagtt cctggagagg ttcaagtccc tgctgcagaa gatgatccat 360

cagcacctgt cctccaggac ccacggctcc gaggactcca ttacatgccc ccctcccatg 420

agcgtggagc acgccgacat ctgggtgaag agctatagcc tctacagccg ggagaggtat 480

atctgtaaca gcggcttcaa gaggaaggcc ggcaccagca gcctcaccga gtgcgtgctg 540

aataaggcta ccaacgtggc tcactggaca acaccctctt taaagtgcat ccggggcggt 600

ggaggatccg gaggaggtgg ctccggcggc ggaggatctg atcccgccgg cctcttggac 660

ctgcggcagg gcatgtttgc gcagctggtg gcccaaaatg ttctgctgat cgatgggccc 720

ctgagctggt acagtgaccc aggcctggca ggcgtgtccc tgacgggggg cctgagctac 780

aaagaggaca cgaaggagct ggtggtggcc aaggctggag tctactatgt cttctttcaa 840

ctagagctgc ggcgcgtggt ggccggcgag ggctcaggct ccgtttcact tgcgctgcac 900

ctgcagccac tgcgctctgc tgctggggcc gccgccctgg ctttgaccgt ggacctgcca 960

cccgcctcct ccgaggctcg gaactcggcc ttcggtttcc agggccgctt gctgcacctg 1020

agtgccggcc agcgcctggg cgtccatctt cacactgagg ccagggcacg ccatgcctgg 1080

cagcttaccc agggcgccac agtcttggga ctcttccggg tgacccccga aatc 1134

<210> 274

<211> 396

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 274

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

20 25 30

Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

35 40 45

Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

50 55 60

Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn

65 70 75 80

Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

85 90 95

Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

100 105 110

Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

115 120 125

Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg

130 135 140

Thr His Gly Ser Glu Asp Ser Ile Thr Cys Pro Pro Pro Met Ser Val

145 150 155 160

Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu

165 170 175

Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser

180 185 190

Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr

195 200 205

Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Asp Pro Ala Gly Leu Leu Asp Leu Arg

225 230 235 240

Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp

245 250 255

Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu

260 265 270

Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala

275 280 285

Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val

290 295 300

Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln

305 310 315 320

Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp

325 330 335

Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln

340 345 350

Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu

355 360 365

His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala

370 375 380

Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile

385 390 395

<210> 275

<211> 1188

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 275

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctcccagggc 60

caggacaggc acatgatccg gatgaggcag ctcatcgaca tcgtcgacca gctgaagaac 120

tacgtgaacg acctggtgcc cgagtttctg cctgcccccg aggacgtgga gaccaactgc 180

gagtggtccg ccttctcctg ctttcagaag gcccagctga agtccgccaa caccggcaac 240

aacgagcgga tcatcaacgt gagcatcaag aagctgaagc ggaagcctcc ctccacaaac 300

gccggcagga ggcagaagca caggctgacc tgccccagct gtgactccta cgagaagaag 360

ccccccaagg agttcctgga gaggttcaag tccctgctgc agaagatgat ccatcagcac 420

ctgtcctcca ggacccacgg ctccgaggac tccattacat gcccccctcc catgagcgtg 480

gagcacgccg acatctgggt gaagagctat agcctctaca gccgggagag gtatatctgt 540

aacagcggct tcaagaggaa ggccggcacc agcagcctca ccgagtgcgt gctgaataag 600

gctaccaacg tggctcactg gacaacaccc tctttaaagt gcatccgggg cggtggagga 660

tccggaggag gtggctccgg cggcggagga tctgatcccg ccggcctctt ggacctgcgg 720

cagggcatgt ttgcgcagct ggtggcccaa aatgttctgc tgatcgatgg gcccctgagc 780

tggtacagtg acccaggcct ggcaggcgtg tccctgacgg ggggcctgag ctacaaagag 840

gacacgaagg agctggtggt ggccaaggct ggagtctact atgtcttctt tcaactagag 900

ctgcggcgcg tggtggccgg cgagggctca ggctccgttt cacttgcgct gcacctgcag 960

ccactgcgct ctgctgctgg ggccgccgcc ctggctttga ccgtggacct gccacccgcc 1020

tcctccgagg ctcggaactc ggccttcggt ttccagggcc gcttgctgca cctgagtgcc 1080

ggccagcgcc tgggcgtcca tcttcacact gaggccaggg cacgccatgc ctggcagctt 1140

acccagggcg ccacagtctt gggactcttc cgggtgaccc ccgaaatc 1188

<210> 276

<400> 276

000

<210> 277

<400> 277

000

<210> 278

<400> 278

000

<210> 279

<400> 279

000

<210> 280

<400> 280

000

<210> 281

<400> 281

000

<210> 282

<400> 282

000

<210> 283

<400> 283

000

<210> 284

<400> 284

000

<210> 285

<400> 285

000

<210> 286

<400> 286

000

<210> 287

<400> 287

000

<210> 288

<400> 288

000

<210> 289

<400> 289

000

<210> 290

<400> 290

000

<210> 291

<400> 291

000

<210> 292

<400> 292

000

<210> 293

<400> 293

000

<210> 294

<400> 294

000

<210> 295

<400> 295

000

<210> 296

<400> 296

000

<210> 297

<400> 297

000

<210> 298

<400> 298

000

<210> 299

<400> 299

000

<210> 300

<211> 485

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 300

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val

145 150 155 160

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

165 170 175

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

180 185 190

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

195 200 205

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

210 215 220

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

225 230 235 240

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

245 250 255

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

260 265 270

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile

275 280 285

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

290 295 300

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

305 310 315 320

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

325 330 335

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

340 345 350

Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile

355 360 365

Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu

370 375 380

Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser

385 390 395 400

Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu

405 410 415

Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser

420 425 430

Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys

435 440 445

Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys

450 455 460

Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His

465 470 475 480

Gly Ser Glu Asp Ser

485

<210> 301

<211> 1455

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 301

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcagcatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgctcc 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga cattacatgc ccccctccca tgagcgtgga gcacgccgac 900

atctgggtga agagctatag cctctacagc cgggagaggt atatctgtaa cagcggcttc 960

aagaggaagg ccggcaccag cagcctcacc gagtgcgtgc tgaataaggc taccaacgtg 1020

gctcactgga caacaccctc tttaaagtgc atccggcagg gccaggacag gcacatgatc 1080

cggatgaggc agctcatcga catcgtcgac cagctgaaga actacgtgaa cgacctggtg 1140

cccgagtttc tgcctgcccc cgaggacgtg gagaccaact gcgagtggtc cgccttctcc 1200

tgctttcaga aggcccagct gaagtccgcc aacaccggca acaacgagcg gatcatcaac 1260

gtgagcatca agaagctgaa gcggaagcct ccctccacaa acgccggcag gaggcagaag 1320

cacaggctga cctgccccag ctgtgactcc tacgagaaga agccccccaa ggagttcctg 1380

gagaggttca agtccctgct gcagaagatg atccatcagc acctgtcctc caggacccac 1440

ggctccgagg actcc 1455

<210> 302

<211> 503

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 302

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

20 25 30

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

35 40 45

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

50 55 60

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

65 70 75 80

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

85 90 95

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

100 105 110

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

115 120 125

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

130 135 140

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys

165 170 175

Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys

180 185 190

Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp

195 200 205

Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu

210 215 220

Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn

225 230 235 240

Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp

245 250 255

Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys

260 265 270

Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu

275 280 285

Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro

290 295 300

Asp Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

305 310 315 320

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

325 330 335

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

340 345 350

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

355 360 365

Ile Arg Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

370 375 380

Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

385 390 395 400

Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

405 410 415

Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn

420 425 430

Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

435 440 445

Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

450 455 460

Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

465 470 475 480

Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg

485 490 495

Thr His Gly Ser Glu Asp Ser

500

<210> 303

<211> 1509

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 303

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccatcccc 60

ccccatgtgc aaaagagcgt gaacaacgat atgatcgtga ccgacaacaa cggcgccgtg 120

aagtttcccc agctctgcaa gttctgcgat gtcaggttca gcacctgcga taatcagaag 180

tcctgcatgt ccaactgcag catcacctcc atctgcgaga agccccaaga agtgtgcgtg 240

gccgtgtggc ggaaaaatga cgagaacatc accctggaga ccgtgtgtca cgaccccaag 300

ctcccttatc acgacttcat tctggaggac gctgcctccc ccaaatgcat catgaaggag 360

aagaagaagc ccggagagac cttctttatg tgttcctgta gcagcgacga gtgtaacgac 420

aacatcatct tcagcgaaga gtacaacacc agcaaccctg atggaggtgg cggatccgga 480

ggtggaggtt ctggtggagg tgggagtatt cctccccacg tgcagaagag cgtgaataat 540

gacatgatcg tgaccgataa caatggcgcc gtgaaatttc cccagctgtg caaattctgc 600

gatgtgaggt tttccacctg cgacaaccag aagtcctgta tgagcaactg ctccatcacc 660

tccatctgtg agaagcctca ggaggtgtgc gtggctgtct ggcggaagaa tgacgagaat 720

atcaccctgg aaaccgtctg ccacgatccc aagctgccct accacgattt catcctggaa 780

gacgccgcca gccctaagtg catcatgaaa gagaaaaaga agcctggcga gacctttttc 840

atgtgctcct gcagcagcga cgaatgcaac gacaatatca tctttagcga ggaatacaat 900

accagcaacc ccgacattac atgcccccct cccatgagcg tggagcacgc cgacatctgg 960

gtgaagagct atagcctcta cagccgggag aggtatatct gtaacagcgg cttcaagagg 1020

aaggccggca ccagcagcct caccgagtgc gtgctgaata aggctaccaa cgtggctcac 1080

tggacaacac cctctttaaa gtgcatccgg cagggccagg acaggcacat gatccggatg 1140

aggcagctca tcgacatcgt cgaccagctg aagaactacg tgaacgacct ggtgcccgag 1200

tttctgcctg cccccgagga cgtggagacc aactgcgagt ggtccgcctt ctcctgcttt 1260

cagaaggccc agctgaagtc cgccaacacc ggcaacaacg agcggatcat caacgtgagc 1320

atcaagaagc tgaagcggaa gcctccctcc acaaacgccg gcaggaggca gaagcacagg 1380

ctgacctgcc ccagctgtga ctcctacgag aagaagcccc ccaaggagtt cctggagagg 1440

ttcaagtccc tgctgcagaa gatgatccat cagcacctgt cctccaggac ccacggctcc 1500

gaggactcc 1509

<210> 304

<400> 304

000

<210> 305

<400> 305

000

<210> 306

<400> 306

000

<210> 307

<400> 307

000

<210> 308

<211> 592

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 308

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val

145 150 155 160

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

165 170 175

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

180 185 190

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

195 200 205

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

210 215 220

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

225 230 235 240

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

245 250 255

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

260 265 270

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile

275 280 285

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

290 295 300

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

305 310 315 320

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

325 330 335

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

340 345 350

Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln Thr

355 360 365

Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala Ser

370 375 380

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly

385 390 395 400

Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser

405 410 415

Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp

420 425 430

Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His Val

435 440 445

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Gly His Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

465 470 475 480

Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg Leu Ser

485 490 495

Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met Ser Trp Val

500 505 510

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile Asn Trp

515 520 525

Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

530 535 540

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

545 550 555 560

Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Ser

565 570 575

Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Arg

580 585 590

<210> 309

<211> 1776

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 309

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcagcatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgctcc 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga cattacatgc ccccctccca tgagcgtgga gcacgccgac 900

atctgggtga agagctatag cctctacagc cgggagaggt atatctgtaa cagcggcttc 960

aagaggaagg ccggcaccag cagcctcacc gagtgcgtgc tgaataaggc taccaacgtg 1020

gctcactgga caacaccctc tttaaagtgc atccggtccg agctgaccca ggaccctgct 1080

gtgtccgtgg ctctgggcca gaccgtgagg atcacctgcc agggcgactc cctgaggtcc 1140

tactacgcct cctggtacca gcagaagccc ggccaggctc ctgtgctggt gatctacggc 1200

aagaacaaca ggccctccgg catccctgac aggttctccg gatcctcctc cggcaacacc 1260

gcctccctga ccatcacagg cgctcaggcc gaggacgagg ctgactacta ctgcaactcc 1320

agggactcct ccggcaacca tgtggtgttc ggcggcggca ccaagctgac cgtgggccat 1380

ggcggcggcg gctccggagg cggcggcagc ggcggaggag gatccgaggt gcagctggtg 1440

gagtccggag gaggagtggt gaggcctgga ggctccctga ggctgagctg tgctgcctcc 1500

ggcttcacct tcgacgacta cggcatgtcc tgggtgaggc aggctcctgg aaagggcctg 1560

gagtgggtgt ccggcatcaa ctggaacggc ggatccaccg gctacgccga ttccgtgaag 1620

ggcaggttca ccatcagcag ggacaacgcc aagaactccc tgtacctgca gatgaactcc 1680

ctgagggccg aggacaccgc cgtgtactac tgcgccaggg gcaggtccct gctgttcgac 1740

tactggggac agggcaccct ggtgaccgtg tccagg 1776

<210> 310

<211> 610

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 310

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

20 25 30

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

35 40 45

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

50 55 60

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

65 70 75 80

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

85 90 95

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

100 105 110

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

115 120 125

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

130 135 140

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys

165 170 175

Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys

180 185 190

Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp

195 200 205

Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu

210 215 220

Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn

225 230 235 240

Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp

245 250 255

Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys

260 265 270

Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu

275 280 285

Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro

290 295 300

Asp Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

305 310 315 320

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

325 330 335

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

340 345 350

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

355 360 365

Ile Arg Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly

370 375 380

Gln Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr

385 390 395 400

Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile

405 410 415

Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly

420 425 430

Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala

435 440 445

Glu Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn

450 455 460

His Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Gly His Gly Gly

465 470 475 480

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

485 490 495

Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly Ser Leu Arg

500 505 510

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr Gly Met Ser

515 520 525

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gly Ile

530 535 540

Asn Trp Asn Gly Gly Ser Thr Gly Tyr Ala Asp Ser Val Lys Gly Arg

545 550 555 560

Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met

565 570 575

Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly

580 585 590

Arg Ser Leu Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

595 600 605

Ser Arg

610

<210> 311

<211> 1830

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 311

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccatcccc 60

ccccatgtgc aaaagagcgt gaacaacgat atgatcgtga ccgacaacaa cggcgccgtg 120

aagtttcccc agctctgcaa gttctgcgat gtcaggttca gcacctgcga taatcagaag 180

tcctgcatgt ccaactgcag catcacctcc atctgcgaga agccccaaga agtgtgcgtg 240

gccgtgtggc ggaaaaatga cgagaacatc accctggaga ccgtgtgtca cgaccccaag 300

ctcccttatc acgacttcat tctggaggac gctgcctccc ccaaatgcat catgaaggag 360

aagaagaagc ccggagagac cttctttatg tgttcctgta gcagcgacga gtgtaacgac 420

aacatcatct tcagcgaaga gtacaacacc agcaaccctg atggaggtgg cggatccgga 480

ggtggaggtt ctggtggagg tgggagtatt cctccccacg tgcagaagag cgtgaataat 540

gacatgatcg tgaccgataa caatggcgcc gtgaaatttc cccagctgtg caaattctgc 600

gatgtgaggt tttccacctg cgacaaccag aagtcctgta tgagcaactg ctccatcacc 660

tccatctgtg agaagcctca ggaggtgtgc gtggctgtct ggcggaagaa tgacgagaat 720

atcaccctgg aaaccgtctg ccacgatccc aagctgccct accacgattt catcctggaa 780

gacgccgcca gccctaagtg catcatgaaa gagaaaaaga agcctggcga gacctttttc 840

atgtgctcct gcagcagcga cgaatgcaac gacaatatca tctttagcga ggaatacaat 900

accagcaacc ccgacattac atgcccccct cccatgagcg tggagcacgc cgacatctgg 960

gtgaagagct atagcctcta cagccgggag aggtatatct gtaacagcgg cttcaagagg 1020

aaggccggca ccagcagcct caccgagtgc gtgctgaata aggctaccaa cgtggctcac 1080

tggacaacac cctctttaaa gtgcatccgg tccgagctga cccaggaccc tgctgtgtcc 1140

gtggctctgg gccagaccgt gaggatcacc tgccagggcg actccctgag gtcctactac 1200

gcctcctggt accagcagaa gcccggccag gctcctgtgc tggtgatcta cggcaagaac 1260

aacaggccct ccggcatccc tgacaggttc tccggatcct cctccggcaa caccgcctcc 1320

ctgaccatca caggcgctca ggccgaggac gaggctgact actactgcaa ctccagggac 1380

tcctccggca accatgtggt gttcggcggc ggcaccaagc tgaccgtggg ccatggcggc 1440

ggcggctccg gaggcggcgg cagcggcgga ggaggatccg aggtgcagct ggtggagtcc 1500

ggaggaggag tggtgaggcc tggaggctcc ctgaggctga gctgtgctgc ctccggcttc 1560

accttcgacg actacggcat gtcctgggtg aggcaggctc ctggaaaggg cctggagtgg 1620

gtgtccggca tcaactggaa cggcggatcc accggctacg ccgattccgt gaagggcagg 1680

ttcaccatca gcagggacaa cgccaagaac tccctgtacc tgcagatgaa ctccctgagg 1740

gccgaggaca ccgccgtgta ctactgcgcc aggggcaggt ccctgctgtt cgactactgg 1800

ggacagggca ccctggtgac cgtgtccagg 1830

<210> 312

<400> 312

000

<210> 313

<400> 313

000

<210> 314

<400> 314

000

<210> 315

<400> 315

000

<210> 316

<211> 551

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 316

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys Ser Val

145 150 155 160

Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro

165 170 175

Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln

180 185 190

Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro

195 200 205

Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr

210 215 220

Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile

225 230 235 240

Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys

245 250 255

Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn

260 265 270

Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Ile

275 280 285

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

290 295 300

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

305 310 315 320

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

325 330 335

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

340 345 350

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg

355 360 365

Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu

370 375 380

Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile

385 390 395 400

Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser

405 410 415

Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val

420 425 430

Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg

435 440 445

Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu

450 455 460

Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val

465 470 475 480

Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe

485 490 495

Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His

500 505 510

Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln Gly

515 520 525

Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly

530 535 540

Leu Pro Ser Pro Arg Ser Glu

545 550

<210> 317

<211> 1653

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 317

atcccccccc atgtgcaaaa gagcgtgaac aacgatatga tcgtgaccga caacaacggc 60

gccgtgaagt ttccccagct ctgcaagttc tgcgatgtca ggttcagcac ctgcgataat 120

cagaagtcct gcatgtccaa ctgcagcatc acctccatct gcgagaagcc ccaagaagtg 180

tgcgtggccg tgtggcggaa aaatgacgag aacatcaccc tggagaccgt gtgtcacgac 240

cccaagctcc cttatcacga cttcattctg gaggacgctg cctcccccaa atgcatcatg 300

aaggagaaga agaagcccgg agagaccttc tttatgtgtt cctgtagcag cgacgagtgt 360

aacgacaaca tcatcttcag cgaagagtac aacaccagca accctgatgg aggtggcgga 420

tccggaggtg gaggttctgg tggaggtggg agtattcctc cccacgtgca gaagagcgtg 480

aataatgaca tgatcgtgac cgataacaat ggcgccgtga aatttcccca gctgtgcaaa 540

ttctgcgatg tgaggttttc cacctgcgac aaccagaagt cctgtatgag caactgctcc 600

atcacctcca tctgtgagaa gcctcaggag gtgtgcgtgg ctgtctggcg gaagaatgac 660

gagaatatca ccctggaaac cgtctgccac gatcccaagc tgccctacca cgatttcatc 720

ctggaagacg ccgccagccc taagtgcatc atgaaagaga aaaagaagcc tggcgagacc 780

tttttcatgt gctcctgcag cagcgacgaa tgcaacgaca atatcatctt tagcgaggaa 840

tacaatacca gcaaccccga cattacatgc ccccctccca tgagcgtgga gcacgccgac 900

atctgggtga agagctatag cctctacagc cgggagaggt atatctgtaa cagcggcttc 960

aagaggaagg ccggcaccag cagcctcacc gagtgcgtgc tgaataaggc taccaacgtg 1020

gctcactgga caacaccctc tttaaagtgc atccggggcg gtggaggatc cggaggaggt 1080

ggctccggcg gcggaggatc tcgcgagggt cccgagcttt cgcccgacga tcccgccggc 1140

ctcttggacc tgcggcaggg catgtttgcg cagctggtgg cccaaaatgt tctgctgatc 1200

gatgggcccc tgagctggta cagtgaccca ggcctggcag gcgtgtccct gacggggggc 1260

ctgagctaca aagaggacac gaaggagctg gtggtggcca aggctggagt ctactatgtc 1320

ttctttcaac tagagctgcg gcgcgtggtg gccggcgagg gctcaggctc cgtttcactt 1380

gcgctgcacc tgcagccact gcgctctgct gctggggccg ccgccctggc tttgaccgtg 1440

gacctgccac ccgcctcctc cgaggctcgg aactcggcct tcggtttcca gggccgcttg 1500

ctgcacctga gtgccggcca gcgcctgggc gtccatcttc acactgaggc cagggcacgc 1560

catgcctggc agcttaccca gggcgccaca gtcttgggac tcttccgggt gacccccgaa 1620

atcccagccg gactcccttc accgaggtcg gaa 1653

<210> 318

<211> 569

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 318

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile

20 25 30

Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe

35 40 45

Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser

50 55 60

Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val

65 70 75 80

Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys

85 90 95

His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala

100 105 110

Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe

115 120 125

Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe

130 135 140

Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp Gly Gly Gly Gly Ser Gly

145 150 155 160

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ile Pro Pro His Val Gln Lys

165 170 175

Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys

180 185 190

Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp

195 200 205

Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu

210 215 220

Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn

225 230 235 240

Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp

245 250 255

Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys

260 265 270

Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu

275 280 285

Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro

290 295 300

Asp Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp

305 310 315 320

Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser

325 330 335

Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu

340 345 350

Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys

355 360 365

Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu

385 390 395 400

Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu

405 410 415

Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly

420 425 430

Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu

435 440 445

Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu

450 455 460

Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu

465 470 475 480

His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu

485 490 495

Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe

500 505 510

Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly

515 520 525

Val His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr

530 535 540

Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro

545 550 555 560

Ala Gly Leu Pro Ser Pro Arg Ser Glu

565

<210> 319

<211> 1707

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 319

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctccatcccc 60

ccccatgtgc aaaagagcgt gaacaacgat atgatcgtga ccgacaacaa cggcgccgtg 120

aagtttcccc agctctgcaa gttctgcgat gtcaggttca gcacctgcga taatcagaag 180

tcctgcatgt ccaactgcag catcacctcc atctgcgaga agccccaaga agtgtgcgtg 240

gccgtgtggc ggaaaaatga cgagaacatc accctggaga ccgtgtgtca cgaccccaag 300

ctcccttatc acgacttcat tctggaggac gctgcctccc ccaaatgcat catgaaggag 360

aagaagaagc ccggagagac cttctttatg tgttcctgta gcagcgacga gtgtaacgac 420

aacatcatct tcagcgaaga gtacaacacc agcaaccctg atggaggtgg cggatccgga 480

ggtggaggtt ctggtggagg tgggagtatt cctccccacg tgcagaagag cgtgaataat 540

gacatgatcg tgaccgataa caatggcgcc gtgaaatttc cccagctgtg caaattctgc 600

gatgtgaggt tttccacctg cgacaaccag aagtcctgta tgagcaactg ctccatcacc 660

tccatctgtg agaagcctca ggaggtgtgc gtggctgtct ggcggaagaa tgacgagaat 720

atcaccctgg aaaccgtctg ccacgatccc aagctgccct accacgattt catcctggaa 780

gacgccgcca gccctaagtg catcatgaaa gagaaaaaga agcctggcga gacctttttc 840

atgtgctcct gcagcagcga cgaatgcaac gacaatatca tctttagcga ggaatacaat 900

accagcaacc ccgacattac atgcccccct cccatgagcg tggagcacgc cgacatctgg 960

gtgaagagct atagcctcta cagccgggag aggtatatct gtaacagcgg cttcaagagg 1020

aaggccggca ccagcagcct caccgagtgc gtgctgaata aggctaccaa cgtggctcac 1080

tggacaacac cctctttaaa gtgcatccgg ggcggtggag gatccggagg aggtggctcc 1140

ggcggcggag gatctcgcga gggtcccgag ctttcgcccg acgatcccgc cggcctcttg 1200

gacctgcggc agggcatgtt tgcgcagctg gtggcccaaa atgttctgct gatcgatggg 1260

cccctgagct ggtacagtga cccaggcctg gcaggcgtgt ccctgacggg gggcctgagc 1320

tacaaagagg acacgaagga gctggtggtg gccaaggctg gagtctacta tgtcttcttt 1380

caactagagc tgcggcgcgt ggtggccggc gagggctcag gctccgtttc acttgcgctg 1440

cacctgcagc cactgcgctc tgctgctggg gccgccgccc tggctttgac cgtggacctg 1500

ccacccgcct cctccgaggc tcggaactcg gccttcggtt tccagggccg cttgctgcac 1560

ctgagtgccg gccagcgcct gggcgtccat cttcacactg aggccagggc acgccatgcc 1620

tggcagctta cccagggcgc cacagtcttg ggactcttcc gggtgacccc cgaaatccca 1680

gccggactcc cttcaccgag gtcggaa 1707

<210> 320

<211> 720

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 320

atgaagtggg tcacatttat ctctttactg ttcctcttct ccagcgccta cagctacttc 60

ggcaaactgg aatccaagct gagcgtgatc cggaatttaa acgaccaagt tctgtttatc 120

gatcaaggta accggcctct gttcgaggac atgaccgact ccgattgccg ggacaatgcc 180

ccccggacca tcttcattat ctccatgtac aaggacagcc agccccgggg catggctgtg 240

acaattagcg tgaagtgtga gaaaatcagc actttatctt gtgagaacaa gatcatctcc 300

tttaaggaaa tgaacccccc cgataacatc aaggacacca agtccgatat catcttcttc 360

cagcggtccg tgcccggtca cgataacaag atgcagttcg aatcctcctc ctacgagggc 420

tactttttag cttgtgaaaa ggagagggat ttattcaagc tgatcctcaa gaaggaggac 480

gagctgggcg atcgttccat catgttcacc gtccaaaacg aggatattac atgcccccct 540

cccatgagcg tggagcacgc cgacatctgg gtgaagagct atagcctcta cagccgggag 600

aggtatatct gtaacagcgg cttcaagagg aaggccggca ccagcagcct caccgagtgc 660

gtgctgaata aggctaccaa cgtggctcac tggacaacac cctctttaaa gtgcatccgg 720

<210> 321

<211> 2607

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 321

atgaaatggg tgacctttat ttctttactg ttcctcttta gcagcgccta ctccatttgg 60

gaactgaaga aggacgtcta cgtggtcgaa ctggactggt atcccgatgc tcccggcgaa 120

atggtggtgc tcacttgtga cacccccgaa gaagacggca tcacttggac cctcgatcag 180

agcagcgagg tgctgggctc cggaaagacc ctcacaatcc aagttaagga gttcggagac 240

gctggccaat acacatgcca caagggaggc gaggtgctca gccattcctt attattatta 300

cacaagaagg aagacggaat ctggtccacc gacattttaa aagatcagaa ggagcccaag 360

aataagacct ttttaaggtg tgaggccaaa aactacagcg gtcgtttcac ttgttggtgg 420

ctgaccacca tttccaccga tttaaccttc tccgtgaaaa gcagccgggg aagctccgac 480

cctcaaggtg tgacatgtgg agccgctacc ctcagcgctg agagggttcg tggcgataac 540

aaggaatacg agtacagcgt ggagtgccaa gaagatagcg cttgtcccgc tgccgaagaa 600

tctttaccca ttgaggtgat ggtggacgcc gtgcacaaac tcaagtacga gaactacacc 660

tcctccttct ttatccggga catcattaag cccgatcctc ctaagaattt acagctgaag 720

cctctcaaaa atagccggca agttgaggtc tcttgggaat atcccgacac ttggagcaca 780

ccccacagct acttctcttt aaccttttgt gtgcaagttc aaggtaaaag caagcgggag 840

aagaaagacc gggtgtttac cgacaaaacc agcgccaccg tcatctgtcg gaagaacgcc 900

tccatcagcg tgagggctca agatcgttat tactccagca gctggtccga gtgggccagc 960

gtgccttgtt ccggcggtgg aggatccgga ggaggtggct ccggcggcgg aggatctcgt 1020

aacctccccg tggctacccc cgatcccgga atgttccctt gtttacacca cagccagaat 1080

ttactgaggg ccgtgagcaa catgctgcag aaagctaggc agactttaga attttaccct 1140

tgcaccagcg aggagatcga ccatgaagat atcaccaagg acaagacatc caccgtggag 1200

gcttgtttac ctctggagct gacaaagaac gagtcttgtc tcaactctcg tgaaaccagc 1260

ttcatcacaa atggctcttg tttagcttcc cggaagacct cctttatgat ggctttatgc 1320

ctcagctcca tctacgagga tttaaagatg taccaagtgg agttcaagac catgaacgcc 1380

aagctgctca tggaccctaa acggcagatc tttttagacc agaacatgct ggctgtgatt 1440

gatgagctga tgcaagcttt aaacttcaac tccgagaccg tccctcagaa gtcctccctc 1500

gaggagcccg atttttacaa gacaaagatc aaactgtgca ttttactcca cgcctttagg 1560

atccgggccg tgaccattga ccgggtcatg agctatttaa acgccagcag cggcacaacc 1620

aacacagtcg ctgcctataa cctcacttgg aagagcacca acttcaaaac catcctcgaa 1680

tgggaaccca aacccgttaa ccaagtttac accgtgcaga tcagcaccaa gtccggcgac 1740

tggaagtcca aatgtttcta taccaccgac accgagtgcg atctcaccga tgagatcgtg 1800

aaagatgtga aacagaccta cctcgcccgg gtgtttagct accccgccgg caatgtggag 1860

agcactggtt ccgctggcga gcctttatac gagaacagcc ccgaatttac cccttacctc 1920

gagaccaatt taggacagcc caccatccaa agctttgagc aagttggcac aaaggtgaat 1980

gtgacagtgg aggacgagcg gactttagtg cggcggaaca acacctttct cagcctccgg 2040

gatgtgttcg gcaaagattt aatctacaca ctgtattact ggaagtcctc ttcctccggc 2100

aagaagacag ctaaaaccaa cacaaacgag tttttaatcg acgtggataa aggcgaaaac 2160

tactgtttca gcgtgcaagc tgtgatcccc tcccggaccg tgaataggaa aagcaccgat 2220

agccccgttg agtgcatggg ccaagaaaag ggcgagttcc gggagaactg ggtgaacgtc 2280

atcagcgatt taaagaagat cgaagattta attcagtcca tgcatatcga cgccacttta 2340

tacacagaat ccgacgtgca cccctcttgt aaggtgaccg ccatgaaatg ttttttactg 2400

gagctgcaag ttatctcttt agagagcgga gacgctagca tccacgacac cgtggagaat 2460

ttaatcattt tagccaataa ctctttatcc agcaacggca acgtgacaga gtccggctgc 2520

aaggagtgcg aagagctgga ggagaagaac atcaaggagt ttctgcaatc ctttgtgcac 2580

attgtccaga tgttcatcaa tacctcc 2607

<210> 322

<211> 240

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 322

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn

20 25 30

Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe

35 40 45

Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile

50 55 60

Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val

65 70 75 80

Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn

85 90 95

Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp

100 105 110

Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp

115 120 125

Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala

130 135 140

Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp

145 150 155 160

Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp Ile

165 170 175

Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys

180 185 190

Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe

195 200 205

Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys

210 215 220

Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg

225 230 235 240

<210> 323

<211> 869

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 323

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp

20 25 30

Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr

35 40 45

Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val

50 55 60

Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp

65 70 75 80

Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser

85 90 95

Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile

100 105 110

Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu

115 120 125

Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile

130 135 140

Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp

145 150 155 160

Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val

165 170 175

Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp

180 185 190

Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val

195 200 205

Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe

210 215 220

Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys

225 230 235 240

Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp

245 250 255

Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln

260 265 270

Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp

275 280 285

Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val

290 295 300

Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser

305 310 315 320

Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

325 330 335

Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe

340 345 350

Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met

355 360 365

Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu

370 375 380

Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu

385 390 395 400

Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser

405 410 415

Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys

420 425 430

Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu

435 440 445

Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met

450 455 460

Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile

465 470 475 480

Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln

485 490 495

Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu

500 505 510

Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg

515 520 525

Val Met Ser Tyr Leu Asn Ala Ser Ser Gly Thr Thr Asn Thr Val Ala

530 535 540

Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu

545 550 555 560

Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr

565 570 575

Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu

580 585 590

Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu

595 600 605

Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser

610 615 620

Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu

625 630 635 640

Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly

645 650 655

Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg

660 665 670

Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile

675 680 685

Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala

690 695 700

Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn

705 710 715 720

Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg

725 730 735

Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu

740 745 750

Phe Arg Glu Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

755 760 765

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

770 775 780

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

785 790 795 800

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

805 810 815

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

820 825 830

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

835 840 845

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

850 855 860

Phe Ile Asn Thr Ser

865

<210> 324

<211> 1452

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 324

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctcccagggc 60

caggacaggc acatgatccg gatgaggcag ctcatcgaca tcgtcgacca gctgaagaac 120

tacgtgaacg acctggtgcc cgagtttctg cctgcccccg aggacgtgga gaccaactgc 180

gagtggtccg ccttctcctg ctttcagaag gcccagctga agtccgccaa caccggcaac 240

aacgagcgga tcatcaacgt gagcatcaag aagctgaagc ggaagcctcc ctccacaaac 300

gccggcagga ggcagaagca caggctgacc tgccccagct gtgactccta cgagaagaag 360

ccccccaagg agttcctgga gaggttcaag tccctgctgc agaagatgat ccatcagcac 420

ctgtcctcca ggacccacgg ctccgaggac tcctccggca ccaccaatac cgtggccgct 480

tataacctca catggaagag caccaacttc gcgacagctc tggaatggga acccaagccc 540

gtcaatcaag tttacaccgt gcagatctcc accaaatccg gagactggaa gagcaagtgc 600

ttctacacaa cagacaccga gtgtgcttta accgacgaaa tcgtcaagga cgtcaagcaa 660

acctatctgg ctcgggtctt ttcctacccc gctggcaatg tcgagtccac cggctccgct 720

ggcgagcctc tctacgagaa ttcccccgaa ttcacccctt atttagagac caatttaggc 780

cagcctacca tccagagctt cgagcaagtt ggcaccaagg tgaacgtcac cgtcgaggat 840

gaaaggactt tagtggcgcg gaataacaca gctttatccc tccgggatgt gttcggcaaa 900

gacctcatct acacactgta ctattggaag tccagctcct ccggcaaaaa gaccgctaag 960

accaacacca acgagttttt aattgacgtg gacaaaggcg agaactactg cttcagcgtg 1020

caagccgtga tcccttctcg taccgtcaac cggaagagca cagattcccc cgttgagtgc 1080

atgggccaag aaaagggcga gttccgggag aactgggtga acgtcatcag cgatttaaag 1140

aagatcgaag atttaattca gtccatgcat atcgacgcca ctttatacac agaatccgac 1200

gtgcacccct cttgtaaggt gaccgccatg aaatgttttt tactggagct gcaagttatc 1260

tctttagaga gcggagacgc tagcatccac gacaccgtgg agaatttaat cattttagcc 1320

aataactctt tatccagcaa cggcaacgtg acagagtccg gctgcaagga gtgcgaagag 1380

ctggaggaga agaacatcaa ggagtttctg caatcctttg tgcacattgt ccagatgttc 1440

atcaatacct cc 1452

<210> 325

<211> 484

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 325

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

20 25 30

Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

35 40 45

Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

50 55 60

Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn

65 70 75 80

Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

85 90 95

Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

100 105 110

Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

115 120 125

Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg

130 135 140

Thr His Gly Ser Glu Asp Ser Ser Gly Thr Thr Asn Thr Val Ala Ala

145 150 155 160

Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Ala Thr Ala Leu Glu Trp

165 170 175

Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys

180 185 190

Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys

195 200 205

Ala Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala

210 215 220

Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala

225 230 235 240

Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu

245 250 255

Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr

260 265 270

Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Ala Arg Asn

275 280 285

Asn Thr Ala Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr

290 295 300

Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys

305 310 315 320

Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr

325 330 335

Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys

340 345 350

Ser Thr Asp Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Glu Phe

355 360 365

Arg Glu Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp

370 375 380

Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp

385 390 395 400

Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu

405 410 415

Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr

420 425 430

Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly

435 440 445

Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys

450 455 460

Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe

465 470 475 480

Ile Asn Thr Ser

<210> 326

<211> 2142

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 326

atgaaatggg tcaccttcat ctctttactg tttttattta gcagcgccta cagcgtgcag 60

ctgcagcagt ccggacccga actggtcaag cccggtgcct ccgtgaaaat gtcttgtaag 120

gcttctggct acacctttac ctcctacgtc atccaatggg tgaagcagaa gcccggtcaa 180

ggtctcgagt ggatcggcag catcaatccc tacaacgatt acaccaagta taacgaaaag 240

tttaagggca aggccactct gacaagcgac aagagctcca ttaccgccta catggagttt 300

tcctctttaa cttctgagga ctccgcttta tactattgcg ctcgttgggg cgatggcaat 360

tattggggcc ggggaactac tttaacagtg agctccggcg gcggcggaag cggaggtgga 420

ggatctggcg gtggaggcag cgacatcgag atgacacagt cccccgctat catgagcgcc 480

tctttaggag aacgtgtgac catgacttgt acagcttcct ccagcgtgag cagctcctat 540

ttccactggt accagcagaa acccggctcc tcccctaaac tgtgtatcta ctccacaagc 600

aatttagcta gcggcgtgcc tcctcgtttt agcggctccg gcagcacctc ttactcttta 660

accattagct ctatggaggc cgaagatgcc gccacatact tttgccatca gtaccaccgg 720

tcccctacct ttggcggagg cacaaagctg gagaccaagc ggagcggcac caccaacaca 780

gtggccgcct acaatctgac ttggaaatcc accaacttca agaccatcct cgagtgggag 840

cccaagcccg ttaatcaagt ttataccgtg cagatttcca ccaagagcgg cgactggaaa 900

tccaagtgct tctataccac agacaccgag tgcgatctca ccgacgagat cgtcaaagac 960

gtgaagcaga catatttagc tagggtgttc tcctaccccg ctggaaacgt ggagagcacc 1020

ggatccgctg gagagccttt atacgagaac tcccccgaat tcacccccta tctggaaacc 1080

aatttaggcc agcccaccat ccagagcttc gaacaagttg gcacaaaggt gaacgtcacc 1140

gtcgaagatg agaggacttt agtgcggagg aacaatacat ttttatcctt acgtgacgtc 1200

ttcggcaagg atttaatcta cacactgtat tactggaagt ctagctcctc cggcaagaag 1260

accgccaaga ccaataccaa cgaattttta attgacgtgg acaagggcga gaactactgc 1320

ttctccgtgc aagctgtgat cccctcccgg acagtgaacc ggaagtccac cgactccccc 1380

gtggagtgca tgggccaaga gaagggagag tttcgtgagc agatcgtgct gacccagtcc 1440

cccgctatta tgagcgctag ccccggtgaa aaggtgacta tgacatgcag cgccagctct 1500

tccgtgagct acatgaactg gtatcagcag aagtccggca ccagccctaa aaggtggatc 1560

tacgacacca gcaagctggc cagcggcgtc cccgctcact ttcggggctc cggctccgga 1620

acaagctact ctctgaccat cagcggcatg gaagccgagg atgccgctac ctattactgt 1680

cagcagtgga gctccaaccc cttcaccttt ggatccggca ccaagctcga gattaatcgt 1740

ggaggcggag gtagcggagg aggcggatcc ggcggtggag gtagccaagt tcagctccag 1800

caaagcggcg ccgaactcgc tcggcccggc gcttccgtga agatgtcttg taaggcctcc 1860

ggctatacct tcacccggta cacaatgcac tgggtcaagc aacggcccgg tcaaggttta 1920

gagtggattg gctatatcaa cccctcccgg ggctatacca actacaacca gaagttcaag 1980

gacaaagcca ccctcaccac cgacaagtcc agcagcaccg cttacatgca gctgagctct 2040

ttaacatccg aggattccgc cgtgtactac tgcgctcggt actacgacga tcattactgc 2100

ctcgattact ggggccaagg taccacctta acagtctcct cc 2142

<210> 327

<211> 714

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 327

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly

20 25 30

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser

35 40 45

Tyr Val Ile Gln Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp

50 55 60

Ile Gly Ser Ile Asn Pro Tyr Asn Asp Tyr Thr Lys Tyr Asn Glu Lys

65 70 75 80

Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ile Thr Ala

85 90 95

Tyr Met Glu Phe Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Tyr

100 105 110

Cys Ala Arg Trp Gly Asp Gly Asn Tyr Trp Gly Arg Gly Thr Thr Leu

115 120 125

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Asp Ile Glu Met Thr Gln Ser Pro Ala Ile Met Ser Ala

145 150 155 160

Ser Leu Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val

165 170 175

Ser Ser Ser Tyr Phe His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro

180 185 190

Lys Leu Cys Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Pro

195 200 205

Arg Phe Ser Gly Ser Gly Ser Thr Ser Tyr Ser Leu Thr Ile Ser Ser

210 215 220

Met Glu Ala Glu Asp Ala Ala Thr Tyr Phe Cys His Gln Tyr His Arg

225 230 235 240

Ser Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Thr Lys Arg Ser Gly

245 250 255

Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn

260 265 270

Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr

275 280 285

Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe

290 295 300

Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp

305 310 315 320

Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn

325 330 335

Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro

340 345 350

Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln

355 360 365

Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu

370 375 380

Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val

385 390 395 400

Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser

405 410 415

Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp

420 425 430

Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro

435 440 445

Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met

450 455 460

Gly Gln Glu Lys Gly Glu Phe Arg Glu Gln Ile Val Leu Thr Gln Ser

465 470 475 480

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

485 490 495

Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

500 505 510

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser

515 520 525

Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser

530 535 540

Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

545 550 555 560

Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu

565 570 575

Glu Ile Asn Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

580 585 590

Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

595 600 605

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

610 615 620

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

625 630 635 640

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

645 650 655

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

660 665 670

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

675 680 685

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp

690 695 700

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

705 710

<210> 328

<211> 17

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Peptide'

<400> 328

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ala

<210> 329

<211> 454

<212> PRT

<213> Chile person

<400> 329

Met Asp Phe Phe Arg Val Val Glu Asn Gln Gln Pro Pro Ala Thr Met

1 5 10 15

Pro Leu Asn Val Ser Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr Asp

20 25 30

Ser Val Gln Pro Tyr Phe Tyr Cys Asp Glu Glu Glu Asn Phe Tyr Gln

35 40 45

Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp Ile

50 55 60

Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser Arg

65 70 75 80

Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Thr Pro Phe Ser

85 90 95

Leu Arg Gly Asp Asn Asp Gly Gly Gly Gly Ser Phe Ser Thr Ala Asp

100 105 110

Gln Leu Glu Met Val Thr Glu Leu Leu Gly Gly Asp Met Val Asn Gln

115 120 125

Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn Ile Ile

130 135 140

Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys Leu Val

145 150 155 160

Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser Gly Ser

165 170 175

Pro Asn Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser Leu Tyr

180 185 190

Leu Gln Asp Leu Ser Ala Ala Ala Ser Glu Cys Ile Asp Pro Ser Val

195 200 205

Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser Cys Ala

210 215 220

Ser Gln Asp Ser Ser Ala Phe Ser Pro Ser Ser Asp Ser Leu Leu Ser

225 230 235 240

Ser Thr Glu Ser Ser Pro Gln Gly Ser Pro Glu Pro Leu Val Leu His

245 250 255

Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln Glu

260 265 270

Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Ala Pro

275 280 285

Gly Lys Arg Ser Glu Ser Gly Ser Pro Ser Ala Gly Gly His Ser Lys

290 295 300

Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr His

305 310 315 320

Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro Ala

325 330 335

Ala Lys Arg Val Lys Leu Asp Ser Val Arg Val Leu Arg Gln Ile Ser

340 345 350

Asn Asn Arg Lys Cys Thr Ser Pro Arg Ser Ser Asp Thr Glu Glu Asn

355 360 365

Val Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn Glu

370 375 380

Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu Glu

385 390 395 400

Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr Ala

405 410 415

Tyr Ile Leu Ser Val Gln Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu

420 425 430

Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln

435 440 445

Leu Arg Asn Ser Cys Ala

450

<210> 330

<211> 1365

<212> DNA

<213> Chile person

<400> 330

ctggattttt ttcgggtagt ggaaaaccag cagcctcccg cgacgatgcc cctcaacgtt 60

agcttcacca acaggaacta tgacctcgac tacgactcgg tgcagccgta tttctactgc 120

gacgaggagg agaacttcta ccagcagcag cagcagagcg agctgcagcc cccggcgccc 180

agcgaggata tctggaagaa attcgagctg ctgcccaccc cgcccctgtc ccctagccgc 240

cgctccgggc tctgctcgcc ctcctacgtt gcggtcacac ccttctccct tcggggagac 300

aacgacggcg gtggcgggag cttctccacg gccgaccagc tggagatggt gaccgagctg 360

ctgggaggag acatggtgaa ccagagtttc atctgcgacc cggacgacga gaccttcatc 420

aaaaacatca tcatccagga ctgtatgtgg agcggcttct cggccgccgc caagctcgtc 480

tcagagaagc tggcctccta ccaggctgcg cgcaaagaca gcggcagccc gaaccccgcc 540

cgcggccaca gcgtctgctc cacctccagc ttgtacctgc aggatctgag cgccgccgcc 600

tcagagtgca tcgacccctc ggtggtcttc ccctaccctc tcaacgacag cagctcgccc 660

aagtcctgcg cctcgcaaga ctccagcgcc ttctctccgt cctcggattc tctgctctcc 720

tcgacggagt cctccccgca gggcagcccc gagcccctgg tgctccatga ggagacaccg 780

cccaccacca gcagcgactc tgaggaggaa caagaagatg aggaagaaat cgatgttgtt 840

tctgtggaaa agaggcaggc tcctggcaaa aggtcagagt ctggatcacc ttctgctgga 900

ggccacagca aacctcctca cagcccactg gtcctcaaga ggtgccacgt ctccacacat 960

cagcacaact acgcagcgcc tccctccact cggaaggact atcctgctgc caagagggtc 1020

aagttggaca gtgtcagagt cctgagacag atcagcaaca accgaaaatg caccagcccc 1080

aggtcctcgg acaccgagga gaatgtcaag aggcgaacac acaacgtctt ggagcgccag 1140

aggaggaacg agctaaaacg gagctttttt gccctgcgtg accagatccc ggagttggaa 1200

aacaatgaaa aggcccccaa ggtagttatc cttaaaaaag ccacagcata catcctgtcc 1260

gtccaagcag aggagcaaaa gctcatttct gaagaggact tgttgcggaa acgacgagaa 1320

cagttgaaac acaaacttga acagctacgg aactcttgtg cgtaa 1365

<210> 331

<211> 1245

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polynucleotide'

<400> 331

atgaagtggg tgaccttcat cagcctgctg ttcctgttct ccagcgccta ctcccagggc 60

caggacaggc acatgatccg gatgaggcag ctcatcgaca tcgtcgacca gctgaagaac 120

tacgtgaacg acctggtgcc cgagtttctg cctgcccccg aggacgtgga gaccaactgc 180

gagtggtccg ccttctcctg ctttcagaag gcccagctga agtccgccaa caccggcaac 240

aacgagcgga tcatcaacgt gagcatcaag aagctgaagc ggaagcctcc ctccacaaac 300

gccggcagga ggcagaagca caggctgacc tgccccagct gtgactccta cgagaagaag 360

ccccccaagg agttcctgga gaggttcaag tccctgctgc agaagatgat ccatcagcac 420

ctgtcctcca ggacccacgg ctccgaggac tccattacat gcccccctcc catgagcgtg 480

gagcacgccg acatctgggt gaagagctat agcctctaca gccgggagag gtatatctgt 540

aacagcggct tcaagaggaa ggccggcacc agcagcctca ccgagtgcgt gctgaataag 600

gctaccaacg tggctcactg gacaacaccc tctttaaagt gcatccgggg cggtggagga 660

tccggaggag gtggctccgg cggcggagga tctcgcgagg gtcccgagct ttcgcccgac 720

gatcccgccg gcctcttgga cctgcggcag ggcatgtttg cgcagctggt ggcccaaaat 780

gttctgctga tcgatgggcc cctgagctgg tacagtgacc caggcctggc aggcgtgtcc 840

ctgacggggg gcctgagcta caaagaggac acgaaggagc tggtggtggc caaggctgga 900

gtctactatg tcttctttca actagagctg cggcgcgtgg tggccggcga gggctcaggc 960

tccgtttcac ttgcgctgca cctgcagcca ctgcgctctg ctgctggggc cgccgccctg 1020

gctttgaccg tggacctgcc acccgcctcc tccgaggctc ggaactcggc cttcggtttc 1080

cagggccgct tgctgcacct gagtgccggc cagcgcctgg gcgtccatct tcacactgag 1140

gccagggcac gccatgcctg gcagcttacc cagggcgcca cagtcttggg actcttccgg 1200

gtgacccccg aaatcccagc cggactccct tcaccgaggt cggaa 1245

<210> 332

<211> 415

<212> PRT

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Polypeptide'

<400> 332

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile

20 25 30

Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu

35 40 45

Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala

50 55 60

Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn

65 70 75 80

Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro

85 90 95

Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro

100 105 110

Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg

115 120 125

Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg

130 135 140

Thr His Gly Ser Glu Asp Ser Ile Thr Cys Pro Pro Pro Met Ser Val

145 150 155 160

Glu His Ala Asp Ile Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu

165 170 175

Arg Tyr Ile Cys Asn Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser

180 185 190

Leu Thr Glu Cys Val Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr

195 200 205

Thr Pro Ser Leu Lys Cys Ile Arg Gly Gly Gly Gly Ser Gly Gly Gly

210 215 220

Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp

225 230 235 240

Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu

245 250 255

Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser

260 265 270

Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys

275 280 285

Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val

290 295 300

Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly

305 310 315 320

Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly

325 330 335

Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu

340 345 350

Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser

355 360 365

Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg

370 375 380

His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg

385 390 395 400

Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu

405 410 415

<210> 333

<211> 15

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Oligonucleotide'

<400> 333

ggtgggtata atggg 15

<210> 334

<211> 25

<212> DNA

<213> artificial sequence

<220>

<221> Source

<223 >/notation= "description of artificial sequence: synthesis

Oligonucleotide'

<400> 334

attattttat tttaaaaaat ttgtg 25

<210> 335

<211> 129

<212> PRT

<213> Chile person

<400> 335

His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser

1 5 10 15

Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile

20 25 30

Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala

35 40 45

Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg

50 55 60

Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile

65 70 75 80

Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu

85 90 95

Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe

100 105 110

Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser

115 120 125

Ser

<210> 336

<211> 126

<212> PRT

<213> Chile person

<400> 336

Gln Gly Cys Pro Thr Leu Ala Gly Ile Leu Asp Ile Asn Phe Leu Ile

1 5 10 15

Asn Lys Met Gln Glu Asp Pro Ala Ser Lys Cys His Cys Ser Ala Asn

20 25 30

Val Thr Ser Cys Leu Cys Leu Gly Ile Pro Ser Asp Asn Cys Thr Arg

35 40 45

Pro Cys Phe Ser Glu Arg Leu Ser Gln Met Thr Asn Thr Thr Met Gln

50 55 60

Thr Arg Tyr Pro Leu Ile Phe Ser Arg Val Lys Lys Ser Val Glu Val

65 70 75 80

Leu Lys Asn Asn Lys Cys Pro Tyr Phe Ser Cys Glu Gln Pro Cys Asn

85 90 95

Gln Thr Thr Ala Gly Asn Ala Leu Thr Phe Leu Lys Ser Leu Leu Glu

100 105 110

Ile Phe Gln Lys Glu Lys Met Arg Gly Met Arg Gly Lys Ile

115 120 125

Claims (194)

1. A method of killing or reducing the number of naturally occurring and/or therapeutically induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

2. A method of reducing naturally occurring and/or treatment-induced accumulation of senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced TGF- β receptor activation.

3. A method of reducing the level of a marker of naturally occurring and/or treatment-induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

4. A method of attenuating the activity of naturally occurring and/or treatment-induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agents that result in attenuation of TGF- β receptor activation.

5. A method of reducing the level and/or activity of one or more SASP factors derived from naturally occurring and/or treatment-induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF- β receptors.

6. The method of any one of claims 1-5, wherein the subject has been diagnosed or identified as having an aging-related disease or inflammatory disease.

7. The method of claim 6, wherein the aging-related disorder is inflammatory aging-related.

8. The method of claim 6, wherein the aging-related disorder is selected from the group consisting of: alzheimer's disease, aneurysms, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, inflammatory bowel disease, intervertebral disc degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, parkinson's disease, age-related loss of elasticity of lung tissue, age-related macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

9. The method of claim 6, wherein the aging-related disorder is selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

10. The method of claim 6, wherein the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, crohn's disease, multiple sclerosis, guillain barre syndrome, psoriasis, grave's disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders and cognitive disorders associated with cancer treatment.

11. The method of any one of claims 1-10, wherein the treatment-induced senescent cells are chemotherapy-induced senescent cells.

12. The method of any one of claims 1-11, wherein the administration of the one or more agents that result in reduced TGF- β receptor activation results in a reduction in the number or activity of naturally occurring senescent cells and/or treatment-induced senescent cells in the subject's target tissue.

13. The method of claim 12, wherein the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vasculature, bone tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

14. A method according to any one of claims 1 to 13, wherein the tgfβ receptor is TGF- β receptor II (TGF- βrii).

15. A method according to any one of claims 1 to 13, wherein the tgfβ receptor is TGF- βriii.

16. The method according to any one of claims 1-15, wherein at least one of the one or more agents that result in reduced activation of a TGF- β receptor is a soluble TGF- β receptor, an extracellular domain of a TGF- β receptor, an antibody that specifically binds TGF- β, an antagonistic antibody that binds TGF- β receptor, an agent that binds LAP, or an agent that binds TGF- β/LAP complex.

17. The method of claim 16, wherein the one or more agents that result in reduced TGF- β receptor activation attenuate activation of TGF- β receptors by binding to LAP or TGF- β/LAP complexes.

18. The method of any one of claims 1-15, wherein at least one of the one or more agents that result in reduced TGF- β receptor activation is a multi-chain chimeric polypeptide comprising:

(e) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(f) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein one or both of the first target binding domain and the second target binding domain specifically bind to a ligand of a TGF- β receptor; or alternatively

One or both of the first target binding domain and the second target binding domain is an antagonistic antigen binding domain that specifically binds to a TGF- β receptor.

19. A method according to claim 18, wherein the TGF- β receptor is TGF- βrii.

20. A method according to claim 18, wherein the TGF- β receptor is TGF- βriii.

21. The method of any one of claims 18-20, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

22. The method of any one of claims 18-20, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

23. The method of any one of claims 18-22, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

24. The method of any one of claims 18-22, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

25. The method of any one of claims 18-24, wherein the second domain and the second target binding domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide.

26. The method of any one of claims 18-24, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

27. The method of any one of claims 18-26, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

28. The method of any one of claims 18-26, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

29. The method of any one of claims 18-28, wherein the first chimeric polypeptide further comprises one or more additional target binding domains.

30. The method of any one of claims 18-29, wherein the second chimeric polypeptide further comprises one or more additional target binding domains.

31. The method of any one of claims 18-30, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

32. The method of claim 31, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93.

33. The method of any one of claims 18-32, wherein the pair of affinity domains are a sushi domain from the alpha chain of the human IL-15 receptor (il15rα) and soluble IL-15.

34. The method of claim 33, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

35. The method of any one of claims 33-34, wherein the soluble IL-15 comprises a mutation that reduces or eliminates IL-15 activity.

36. The method of any one of claims 18-32, wherein the pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, and SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

37. The method of any one of claims 18-32, wherein the first domain or the second domain of the pair of affinity domains is a soluble common gamma chain family cytokine or an antigen binding domain that specifically binds a common gamma chain family cytokine receptor.

38. The method of any one of claims 18-37, wherein the first target binding domain and the second target binding domain comprise a soluble TGF- β receptor.

39. A method according to claim 38, wherein the soluble TGF- β receptor is a soluble TGF- βrii.

40. A method according to claim 39, wherein the soluble TGF-beta RII comprises a first sequence at least 80% identical to SEQ ID NO. 183 and a second sequence at least 80% identical to SEQ ID NO. 183, wherein the first and second sequences are separated by a linker.

41. A method according to claim 40, wherein the soluble TGF-beta RII comprises a first sequence at least 90% identical to SEQ ID NO. 183 and a second sequence at least 90% identical to SEQ ID NO. 183.

42. A method according to claim 41, wherein the soluble TGF- β RII comprises a first sequence of SEQ ID NO. 183 and a second sequence of SEQ ID NO. 183.

43. The method of claim 40, wherein the linker comprises the sequence of SEQ ID NO. 102.

44. A method according to claim 39, wherein the soluble TGF- β RII comprises a sequence at least 80% identical to SEQ ID NO 188.

45. A method according to claim 44, wherein the soluble TGF- β RII comprises a sequence at least 90% identical to SEQ ID NO 188.

46. A method according to claim 45, wherein the soluble TGF- β RII comprises the sequence of SEQ ID NO. 188.

47. The method of claim 18, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 236.

48. The method of claim 47, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO. 236.

49. The method of claim 48, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO. 236.

50. The method of claim 18, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID No. 193.

51. The method of claim 50, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO. 236.

52. The method of claim 51, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO 193.

53. The method of claim 52, wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO. 193.

54. The method of claim 53, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO. 236.

55. The method of any one of claims 1-15, wherein at least one of the one or more agents that result in reduced TGF- β receptor activation is a single chain chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A second target binding domain that is complementary to the first target binding domain,

wherein one or both of the first target binding domain and the second target binding domain specifically bind to a ligand of a TGF- β receptor; or alternatively

One or both of the first target binding domain and the second target binding domain is an antagonistic antigen binding domain that specifically binds to a TGF- β receptor.

56. A method according to claim 55, wherein the TGF- β receptor is TGF- βRII.

57. A method according to claim 55, wherein the TGF- β receptor is TGF- βriii.

58. The method of any one of claims 55-57, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other.

59. The method of any one of claims 55-57, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain.

60. The method of any one of claims 55-59, wherein the soluble tissue factor domain and the second target binding domain are directly adjacent to each other.

61. The method of any one of claims 55-59, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain.

62. The method of any one of claims 55-61, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

63. The method of any one of claims 55-61, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

64. The method of any one of claims 55-63, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

65. The method of claim 64, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO. 93.

66. The method of any one of claims 55 to 65, wherein the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-and/or C-terminus.

67. The method of any one of claims 55-56, wherein the first target binding domain and the second target binding domain comprise a soluble TGF- β receptor.

68. A method according to claim 67, wherein the soluble TGF- β receptor is a soluble TGF- βRII.

69. A method according to claim 68, wherein said soluble TGF- βrii comprises a first sequence at least 80% identical to SEQ ID No. 183 and a second sequence at least 80% identical to SEQ ID No. 183, wherein said first and second sequences are separated by a linker.

70. The method of claim 69, wherein the soluble TGF-beta RII comprises a first sequence at least 90% identical to SEQ ID NO. 183 and a second sequence at least 90% identical to SEQ ID NO. 183.

71. A method according to claim 70, wherein the soluble TGF- βrii comprises a first sequence of SEQ ID No. 183 and a second sequence of SEQ ID No. 183.

72. The method of claim 69, wherein the linker comprises the sequence of SEQ ID NO. 102.

73. A method as claimed in claim 72, wherein said soluble TGF- βrii comprises a sequence at least 80% identical to SEQ ID No. 188.

74. A method according to claim 73, wherein said soluble TGF- βrii comprises a sequence at least 90% identical to SEQ ID No. 188.

75. A method according to claim 74, wherein said soluble TGF- βrii comprises the sequence of SEQ ID No. 188.

76. The method of any one of claims 1-75, wherein the method comprises administering two or more doses of the one or more agents that result in reduced activation of TGF- β receptors to the subject.

77. The method of claim 76, wherein any two consecutive doses of the two or more doses are administered between about 1 week and about one year apart.

78. The method of claim 77, wherein any consecutive two of said two or more doses are administered between about 1 week and about 6 months apart.

79. The method of claim 78, wherein any consecutive two of the two or more doses are administered between about 1 week and about 2 months apart.

80. The method of claim 79, wherein any two consecutive doses of the two or more doses are administered between about 1 week and about 1 month apart.

81. The method of any one of claims 76-80, wherein the two or more doses are administered by subcutaneous administration.

82. The method of any one of claims 76-80, wherein the two or more doses are administered by intramuscular injection.

83. The method of any one of claims 76-82, wherein the two or more doses are administered over a period of about 1 year to about 60 years.

84. The method of claim 83, wherein the two or more doses are administered over a period of about 1 year to about 50 years.

85. The method of claim 84, wherein the two or more doses are administered over a period of about 1 year to about 40 years.

86. The method of claim 85, wherein the two or more doses are administered over a period of about 1 year to about 30 years.

87. The method of claim 86, wherein the two or more doses are administered over a period of about 1 year to about 20 years.

88. The method of claim 87, wherein the two or more doses are administered over a period of about 1 year to about 10 years.

89. The method of any one of claims 1-88, wherein the one or more agents that result in reduced TGF- β receptor activation of a first agent begin when the subject reaches at least 30 years of age.

90. The method of claim 89, wherein the one or more agents that result in reduced TGF- β receptor activation of the first agent begin when the subject reaches at least 40 years of age.

91. The method of claim 90, wherein the one or more agents that result in reduced TGF- β receptor activation of the first agent begin by the time the subject reaches at least 50 years of age.

92. The method of claim 91, wherein the one or more agents that result in reduced TGF- β receptor activation of the first agent begin by the time the subject reaches at least 60 years of age.

93. The method of any one of claims 1-92, wherein each of the two or more doses is administered at a dose of about 0.01mg/kg of each agent that results in reduced TGF- β receptor activation to about 10mg/kg of each agent that results in reduced TGF- β receptor activation.

94. The method of claim 93, wherein each of the two or more doses is administered at a dose of about 0.02mg/kg of each agent that results in reduced TGF- β receptor activation to about 5mg/kg of each agent that results in reduced TGF- β receptor activation.

95. The method of any one of claims 1-3 and 9-94, wherein the subject has not been diagnosed or identified as having an aging-related or inflammatory disease.

96. The method of any one of claims 1-3 and 9-95, wherein the subject has not been previously treated with a chemotherapeutic agent.

97. The method of any one of claims 1-3 and 9-95, wherein the subject has not been previously treated with a therapeutic agent that induces cellular senescence.

98. A method of killing or reducing the number of naturally occurring and/or therapeutically induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

99. A method of reducing accumulation of naturally occurring and/or treatment-induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

100. A method of reducing the level of a marker of naturally occurring and/or treatment-induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

101. A method of attenuating the activity of naturally occurring and/or treatment-induced senescent cells in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

102. A method of reducing the level or activity of naturally occurring and/or therapeutically induced senescent cell derived SASP factor in a subject, the method comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators.

103. The method of any one of claims 98-102, wherein the subject has been diagnosed with or identified as having an aging-related or inflammatory disease.

104. The method of claim 103, wherein the aging-related disorder is inflammatory aging-related.

105. The method of claim 103, wherein the aging-related disorder is selected from the group consisting of: alzheimer's disease, aneurysms, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, inflammatory bowel disease, intervertebral disc degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, loss of bone mass, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, parkinson's disease, age-related loss of elasticity of lung tissue, age-related macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, blood brain barrier damage, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

106. The method of claim 103, wherein the aging-related disorder is selected from the group consisting of: solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, ewing's sarcoma, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-hodgkin's lymphomas, chronic Lymphocytic Leukemia (CLL), acute Myelogenous Leukemia (AML), chronic Myelogenous Leukemia (CML), acute Lymphocytic Leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

107. The method of claim 103, wherein the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, crohn's disease, multiple sclerosis, guillain barre syndrome, psoriasis, grave's disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders and cognitive disorders associated with cancer treatment.

108. The method of any one of claims 98-107, wherein the treatment-induced senescent cells are chemotherapy-induced senescent cells.

109. The method of any one of claims 98-107, wherein the administration of the one or more common gamma chain family cytokine receptor activators results in a reduction in the number of naturally occurring senescent cells and/or treatment-induced senescent cells in a target tissue of the subject.

110. The method of claim 109, wherein the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature, bone tissue, central Nervous System (CNS) tissue, ocular tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue.

111. The method of any one of claims 98-110, wherein at least one of the one or more common gamma chain family cytokine receptor activators is a complex of a common gamma chain family cytokine or a functional fragment thereof with an antibody or antibody fragment that specifically binds to the common gamma chain family cytokine or a functional fragment thereof.

112. The method of any one of claims 98-110, wherein at least one of the one or more common gamma chain family cytokine receptor activators is a single chain chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A second target binding domain that is complementary to the first target binding domain,

wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine, an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common gamma chain family cytokine receptor, or an antigen binding domain that specifically binds to a common gamma chain family cytokine.

113. The method of claim 112, wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine.

114. The method of claim 113, wherein the soluble common gamma chain family cytokine is selected from the group consisting of: soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15 and soluble IL-21.

115. The method of claim 114, wherein one or both of the first and second target binding domains comprises an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor.

116. The method of claim 115, wherein the common gamma chain family cytokine receptor is a receptor for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

117. The method of claim 115 or 116, wherein the agonistic antigen binding domain is a scFv, VHH, or VNAR.

118. The method of any one of claims 112-117, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other.

119. The method of any one of claims 112-117, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain.

120. The method of any one of claims 112-119, wherein the soluble tissue factor domain and the second target binding domain are directly adjacent to each other.

121. The method of any one of claims 112-119, wherein the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain.

122. The method of any one of claims 208 to 218, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

123. The method of claim 122, wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

124. The method of claim 123, wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

125. The method of any one of claims 112-121, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

126. The method of any one of claims 112-125, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

127. The method of claim 126, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93.

128. The method of any one of claims 112 to 127, wherein the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-and/or C-terminus.

129. The method of any one of claims 98-110, wherein at least one of the one or more common gamma chain family cytokine receptor activators is a multi-chain chimeric polypeptide comprising:

(a) A first chimeric polypeptide comprising:

(i) A first target binding domain;

(ii) A soluble tissue factor domain; and

(iii) A first domain of a pair of affinity domains;

(b) A second chimeric polypeptide comprising:

(i) A second domain of the pair of affinity domains; and

(ii) A second target binding domain that is complementary to the first target binding domain,

wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine, an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common gamma chain family cytokine receptor, or an antigen binding domain that specifically binds to a common gamma chain family cytokine.

130. The method of claim 129, wherein one or both of the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine.

131. The method of claim 130, wherein the soluble common gamma chain family cytokine is selected from the group consisting of: soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15 and soluble IL-21.

132. The method of claim 129, wherein one or both of the first target binding domain and the second target binding domain comprises an agonistic antigen binding domain that specifically binds to a common gamma chain family cytokine receptor.

133. The method of claim 132, wherein the common gamma chain family cytokine receptor is a receptor for one or more of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

134. The method of claim 132 or 133, wherein the agonistic antigen binding domain is a scFv, VHH, or VNAR.

135. The method of any one of claims 129-134, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

136. The method of any of claims 129-134, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

137. The method of any one of claims 129-136, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

138. The method of any of claims 129-136, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain in the pair of affinity domains.

139. The method of any one of claims 129-138, wherein the second domain and the second target binding domain of the pair of affinity domains directly abut each other in the second chimeric polypeptide.

140. The method of any of claims 129-138, wherein a second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains in the second chimeric polypeptide and the second target binding domain.

141. The method of any of claims 129-140, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

142. The method of any of claims 129-140, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

143. The method of any of claims 129-142, wherein the first chimeric polypeptide further comprises one or more additional target binding domains.

144. The method of any of claims 129-143, wherein the second chimeric polypeptide further comprises one or more additional target binding domains.

145. The method of any of claims 129-144, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

146. The method of claim 145, wherein the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID No. 93.

147. The method of any of claims 129-146, wherein the pair of affinity domains are a sushi domain from an alpha chain of a human IL-15 receptor (IL 15 ra) and soluble IL-15.

148. The method of any one of claims 129-146, wherein the pair of affinity domains is selected from the group consisting of: bacillus ribonuclease and bacillus ribonuclease inhibitors, PKA and AKAP, adaptor/docking tag modules based on mutant ribonuclease I fragments, and SNARE modules based on the interaction of protein synaptotagmin, synaptotagmin and SNAP 25.

149. The method of any of claims 129-146, wherein the first domain or the second domain of the pair of affinity domains is a soluble common gamma chain family cytokine or an antigen binding domain that specifically binds a common gamma chain family cytokine receptor.

150. The method of any one of claims 98-110, wherein at least one of the one or more common gamma chain family cytokine receptor activators is a soluble IL-15 or IL-15 agonist.

151. The method of claim 150, wherein the soluble IL-15 is at least 90% identical to SEQ ID No. 82.

152. The method of claim 150, wherein the IL-15 agonist comprises a complex of IL-15 and all or part of a soluble IL-15 receptor (IL-15R).

153. The method of claim 152, wherein the partially soluble IL-15R is part of IL-15 ra.

154. The method of claim 153, wherein the partially soluble IL-15 ra is the sushi domain of IL-15 ra.

155. The method of any of claims 152-154, wherein the IL-15 agonist further comprises an Fc domain.

156. The method of claim 150, wherein the IL-15 agonist comprises a fusion protein comprising IL-15 and sushi domain from IL-15 ra.

157. The method of any one of claims 98-110, wherein one of the one or more common gamma chain family cytokine receptor activators is a soluble IL-2 or IL-2 agonist.

158. The method of any one of claims 98-110, wherein one of the one or more common gamma chain family cytokine receptor activators is an antibody or antigen-binding antibody fragment that specifically binds a common gamma chain family cytokine.

159. The method of any one of claims 98-158, wherein the method comprises administering to the subject one, two, or more doses of the one or more common gamma chain family cytokine receptor activators.

160. The method of claim 159, wherein any consecutive two of the two or more doses are administered between about 1 week and about one year apart.

161. The method of claim 160, wherein any two consecutive doses of the two or more doses are administered between about 1 week and about 6 months apart.

162. The method of claim 161, wherein any consecutive two of the two or more doses are administered between about 1 week and about 2 months apart.

163. The method of claim 162, wherein any two consecutive doses of the two or more doses are administered between about 1 week and about 1 month apart.

164. The method of any of claims 159-163, wherein the one, two, or more doses are administered by subcutaneous administration.

165. The method of any of claims 159-163, wherein the two or more doses are administered by intramuscular injection.

166. The method of any of claims 159-165, wherein the two or more doses are administered over a period of about 1 year to about 60 years.

167. The method of claim 166, wherein the two or more doses are administered over a period of about 1 year to about 50 years.

168. The method of claim 167, wherein the two or more doses are administered over a period of about 1 year to about 40 years.

169. The method of claim 168, wherein the two or more doses are administered over a period of about 1 year to about 30 years.

170. The method of claim 169, wherein the two or more doses are administered over a period of about 1 year to about 20 years.

171. The method of claim 170, wherein the two or more doses are administered over a period of about 1 year to about 10 years.

172. The method of any one of claims 98-171, wherein each of the two or more doses is administered at a dose of about 0.01mg/kg of each common gamma chain family cytokine receptor activator to about 10mg/kg of each common gamma chain family cytokine receptor activator.

173. The method of claim 171, wherein each of the two or more doses is administered at a dose of about 0.02mg/kg of each common gamma chain family cytokine receptor activator to about 5mg/kg of each common gamma chain family cytokine receptor activator.

174. The method of any one of claims 98-173, wherein the first dose of the one or more common gamma chain family cytokine receptor activators begins when a subject reaches at least 30 years of age.

175. The method of claim 174, wherein the first dose of the one or more common gamma chain family cytokine receptor activators begins when a subject reaches at least 40 years of age.

176. The method of claim 175, wherein the first dose of the one or more common gamma chain family cytokine receptor activators begins when a subject reaches at least 50 years old.

177. The method of claim 176, wherein the first dose of the one or more common gamma chain family cytokine receptor activators begins when the subject reaches at least 60 years of age.

178. The method of any of claims 98-102 and 108-177, wherein the subject has not been diagnosed with or identified as having an aging-related or inflammatory disease.

179. The method of any one of claims 98-102 and 98-102, wherein the subject has not been previously treated with a chemotherapeutic agent.

180. The method of any one of claims 98-102 and 98-102, wherein the subject has not been previously treated with a therapeutic agent that induces cellular senescence.

181. The method of any one of claims 98-180, wherein the method further comprises administering to the subject at least one or more agents that result in reduced TGF- β receptor activation.

182. The method of claim 181, wherein at least one of the one or more agents that result in reduced activation of a TGF- β receptor is a soluble TGF- β receptor, an extracellular domain of a TGF- β receptor, an antibody that specifically binds TGF- β, an antagonistic antibody that binds TGF- β receptor, an agent that binds LAP, or an agent that binds TGF- β/LAP complex.

183. The method of claim 182, wherein the one or more agents that result in reduced TGF- β activation attenuate activation of TGF- β receptors by binding to LAP or TGF- β/LAP complex.

184. The method of any one of claims 31, 32, 64, 65, 126, 127, 145, and 146, wherein the soluble human tissue factor domain does not trigger blood clotting.

185. The method of any one of claims 1-184, wherein the method further comprises administering an additional therapeutic agent selected from the group consisting of: combinations of agents, such as checkpoint inhibitors, chemotherapeutic agents and therapeutic antibodies.

186. The method of any one of claims 55-75, wherein the single-chain chimeric polypeptide is stable in human serum at 37 ℃ for at least 10 days.

187. The method of any one of claims 18-54, wherein the multi-chain chimeric polypeptide is stable in human serum at 37 ℃ for at least 10 days.

188. The method of any one of claims 112-128, wherein the single-chain chimeric polypeptide does not have significant clotting activity.

189. The method of any of claims 129-149, wherein the multi-chain chimeric polypeptide does not have significant clotting activity.

190. The method of any one of claims 1-189, wherein the method results in regeneration of aged immune cells in the subject.

191. The method of claim 190, wherein the regeneration of the aged immune cells results in a reduction in the number of diseased cells or infectious agents in the subject.

192. The method of claim 190 or 191, wherein the aged immune cells comprise one or more of aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils, aged basophils, aged eosinophils, aged kupfer cells, and aged microglia cells.

193. The method of claim 191, where the diseased cells include cancer cells, virally-infected cells, and intra-cellular bacterially-infected cells.

194. The method of claim 191, where the infectious agent comprises a virus, a bacterium, a fungus, and a parasite.