JP2023527869A - Treatments for age-related disorders - Google Patents

Abstract

Killing or reducing the number of naturally occurring and/or treatment-induced senescent and diseased cells in a subject in need thereof, naturally occurring and/or treatment-induced in a subject in need thereof Provided herein are methods of reducing the accumulation of senescent and diseased cells, said methods comprising reducing activation of one or more common gamma chain family cytokine receptor activators and/or TGF-β receptors. administering to the subject a therapeutically effective amount of one or more agents that provide
[Selection drawing] Fig. 263

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is filed June 1, 2020, U.S. Provisional Patent Application No. 63/032,933, filed June 1, 2020, which is hereby incorporated by reference in its entirety. International Patent Application No. PCT/US2020/035598, filed Nov. 25, 2020, and U.S. Provisional Patent Application No. 63/118,536, filed Nov. 25, 2020.

TECHNICAL FIELD This disclosure relates to the fields of immunology and cell biology.

BACKGROUND Senescence is a form of irreversible growth arrest that involves phenotypic changes, resistance to apoptosis, and activation of injury-sensing signaling pathways. Cellular senescence was first described in cultured human fibroblasts that lost the ability to proliferate, reaching permanent arrest after approximately 50 population doublings (termed the Hayflick limit). Aging is viewed as a stress response that can be induced by a wide range of endogenous and exogenous insults, including oxidative and genotoxic stress, DNA damage, telomere attrition, oncogenic activation, mitochondrial dysfunction, or chemotherapeutic agents. .

Senescent cells remain metabolically active and can influence tissue hemostasis, disease and aging through their secretory phenotype. Aging is regarded as a physiological process that is important in promoting wound healing, tissue homeostasis, regeneration, and regulation of fibrosis. For example, transient induction of senescent cells is observed during healing and contributes to wound resolution. Aging also plays a role in tumor suppression. Accumulation of senescent cells also promotes aging and age-related diseases and conditions. The senescent phenotype may also induce chronic inflammatory responses, thereby enhancing the chronic inflammatory state and promoting tumor growth. The link between senescence and aging was originally based on the observation that senescent cells accumulate in aging tissue. The use of transgenic models has made it possible to systematically detect senescent cells in many age-related disorders. Strategies to selectively eliminate senescent cells demonstrate that senescent cells can play a causal role in age-related disorders.

Cellular senescence is a series of progressive and phenotypically diverse cellular states acquired after initial growth arrest (van Deursen, Nature 509(7501):439-446, 2014). Senescent cells are therefore a heterogeneous cell population with few shared nuclear properties (Dou et al., Nature 550(7676):402-406, 2017). Therefore, it is difficult to identify targets for common senolytic drugs. This further hinders the goal of developing senolytic agents that selectively, safely and effectively eliminate senescent cells by systemic administration. As described above, immune cells are effector cells for the natural elimination of senescent cells after they have fulfilled their physiological roles (Brighton et al., Elife 6, 2017 (Non-Patent Document 3)). . Weakening of the immune system during the aging process allows senescent cells to accumulate (Karin et al., Nat. Comm. 10(1):5495, 2019, Chambers et al. , J. Allergy Clin. Immunol.145(5):1323-1331, 2020 (Non-Patent Document 5)).

van Deursen, Nature 509(7501):439-446, 2014 Dou et al. , Nature 550(7676): 402-406, 2017 Brighton et al. , Elife 6, 2017 Karin et al. , Nat. Comm. 10(1):5495, 2019 Chambers et al. , J. Allergy Clin. Immunol. 145(5): 1323-1331, 2020

SUMMARY The present invention provides agents in mammals that result in decreased activation of TGF-beta receptors or common gamma chain family cytokine receptor activators (e.g., complexes of gamma chain cytokines and their cognate receptors). It is based on the discovery that subcutaneous administration of AG stimulates and activates immune cells to restore the ability to effectively, selectively and safely reduce senescent cells in vivo. In view of this discovery, spontaneous 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 decreased activation of TGF-β receptors. Provided herein are methods of killing cells or reducing the number of senescent cells. Also a method of reducing spontaneous 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. provided herein. Reducing levels of markers of spontaneous and/or treatment-induced senescence in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF-beta receptors Methods are also provided herein. Also a method of reducing spontaneous and/or treatment-induced senescent cell activity in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF-beta receptors. provided herein. one or more treatments derived from 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 decreased activation of TGF-β receptors. Also provided herein are methods of reducing the level and/or activity of senescence-associated secretory phenotype (“SASP”) factors.

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

The present invention also provides that administration of NK cell activators to mammals with cancer results in tumor inhibition, administration of NK cell activators to diabetic animal models improves the appearance and texture of skin and hair, and It is based on the finding that it showed a reduction in blood sugar levels. In view of this discovery provided herein, a method of treating an age-related disease or condition in a subject in need thereof comprises: A method comprising administering to a subject identified as a therapeutically effective amount of one or more natural killer (NK) cell activators and/or a therapeutically effective number of activated NK cells is described herein provided to A method of killing senescent cells or reducing the number of senescent cells in a subject in need of killing senescent cells or reducing the number of senescent cells, said subject comprising a therapeutically effective number of one or more natural killers ( Also provided herein are methods comprising administering a therapeutically effective amount of a NK) cell activator and/or activated NK cells. 1. A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time, comprising administering to said subject a therapeutically effective number of 1 Also provided herein are methods comprising administering therapeutically effective amounts of one or more natural killer (NK) cell activators and/or activated NK cells. A method of helping treat obesity in a subject in need of help treating obesity 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 comprising administering and/or activated NK cells.

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

Also methods of reducing spontaneous 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. provided herein.

Reducing levels of spontaneous and/or treatment-induced markers 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-beta receptors A method is also provided herein.

Also a method of reducing spontaneous and/or treatment-induced senescent cell activity 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. provided herein.

one or more treatments derived from 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 decreased activation of TGF-β receptors. Also provided herein are methods of reducing the level and/or activity of SASP factors.

In some embodiments of any of the methods described herein, the subject has been previously diagnosed or identified as having an age-related disease or an inflammatory disease. In some embodiments of any of the methods described herein, the age-related disease is inflammatory senescence-related. In some embodiments of any of the methods described herein, the age-related disease is Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, inflammatory bowel disease disease, disc degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, Sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related pulmonary tissue elastic loss, age-related macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, muscle selected from the group of amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, susceptibility to infection, chronic inflammation, and renal dysfunction . In some embodiments of any of the methods described herein, the age-related disease is solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, Rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) , chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, 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, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer cancer, and hepatocellular carcinoma. In some embodiments of any of the methods described herein, the inflammatory disease is 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-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, nonalcoholic steatohepatitis, mood disorders, and cancer treatment-related cognition Selected from a group of disorders.

In some embodiments of any of the methods described herein, the treatment-induced senescent cells are chemotherapy-induced senescent cells. In some embodiments of any of the methods described herein, administration of one or more agents that result in decreased activation of TGF-β receptors is associated with spontaneous aging of target tissues in the subject. resulting in a decrease in the number or activity of cells and/or treatment-induced senescent cells. In some embodiments of any of the methods described herein, the target tissue is adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vasculature, bone tissue, central nervous system tissue. 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 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 decreased TGF-β receptor activation is soluble TGF-β an agent that binds to a receptor, an extracellular domain of a TGF-beta receptor, an antibody that specifically binds to TGF-beta, an antagonistic antibody that binds to a TGF-beta receptor, a latent associated peptide ("LAP"), or An agent that binds to the TGF-β/LAP complex. In some embodiments of any of the methods described herein, the one or more agents that result in decreased activation of TGF-β receptors are to LAP or to TGF-β/LAP complexes. It reduces TGF-β receptor activation through binding to the body.

In some embodiments of any of the methods described herein, at least one of the one or more agents that result in decreased activation of the TGF-β receptor comprises (a) ( a first chimeric polypeptide comprising a first of i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a pair of affinity domains; and (b) (i) an affinity pair and (ii) a second chimeric polypeptide comprising a second target binding domain, wherein the first target binding domain and the second target binding domain. Either one or both of the domains specifically bind to a ligand of the TGF-beta receptor, or one or both of the first target binding domain and the second target binding domain bind to the TGF-beta receptor. It is an antagonist antigen-binding domain that specifically binds to the body. In some embodiments of any of the methods described herein, the TGF-β receptor is TGFβRII. In some embodiments of any of the methods described herein, the TGF-β receptor is TGFβRIII.

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 within the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide has a further includes a linker sequence.

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

In some embodiments of any of the methods described herein, the second domain and second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. are doing. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises the second domain and the second of the pair of affinity domains within the second chimeric polypeptide. It further comprises a linker sequence between it and the 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 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 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 that is at least 80% identical to SEQ ID NO:93.

In some embodiments of any of the methods described herein, the pair of affinity domains is a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) and a soluble IL-15 be. 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 mutations to reduce or eliminate IL-15 activity.

In some embodiments of any of the methods described herein, the pair of affinity domains are barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments. , and the group of SNARE modules based on the interaction of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. 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 a common gamma chain family cytokine An antigen-binding domain that specifically binds to a receptor.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain comprise a soluble TGF-beta receptor. In some embodiments of any of the methods described herein, the soluble TGF-β receptor is soluble TGFβRII. In some embodiments of any of the methods described herein, the soluble TGFβRII is a first sequence that is at least 80% identical to SEQ ID NO: 183 and at least 80% identical to SEQ ID NO: 183 A second sequence, wherein the first and second sequences are separated by a linker. In some embodiments of any of the methods described herein, the soluble TGFβRII is a first sequence that is at least 90% identical to SEQ ID NO: 183 and at least 90% identical to SEQ ID NO: 183 Contains a second array. In some embodiments of any of the methods described herein, the soluble TGFβRII comprises the first sequence of SEQ ID NO:183 and the 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 TGFβRII comprises a sequence that is at least 80% identical to SEQ ID NO:188. In some embodiments of any of the methods described herein, the soluble TGFβRII comprises a sequence that is at least 90% identical to SEQ ID NO:188. In some embodiments of any of the methods described herein, the soluble TGF-βRII 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 that is 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 that is 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 that is 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 that is 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 that is 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 decreased TGF-β receptor activation is (i) a second A single chain chimeric polypeptide comprising one target binding domain, (ii) a soluble tissue factor domain, and (iii) a second target binding domain, wherein specifically binds to a ligand of the TGF-beta receptor, or one or both of the first target binding domain and the second target binding domain are specific for the TGF-beta receptor It is an antagonist antigen-binding domain that specifically binds. In some embodiments of any of the methods described herein, the TGF-β receptor is TGF-βRII. In some embodiments of any of the methods described herein, the TGF-β receptor is TGFβRIII.

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

In some embodiments of any of the methods described herein, the method is directed to two or more doses of one or more agents that result in decreased activation of the TGF-β receptor. including administering. In some embodiments of any of the methods described herein, any two consecutive doses of the two or more doses are administered about one week to about one year apart. In some embodiments of any of the methods described herein, any two consecutive doses of the two or more doses are administered about one week to about six months apart. In some embodiments of any of the methods described herein, any two consecutive doses of the two or more doses are administered about one week to about two months apart. In some embodiments of any of the methods described herein, any two consecutive doses of the two or more doses are administered about one week to about one month apart.

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

In some embodiments of any of the methods described herein, the 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, the 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, the 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, the 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, the 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, the 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 first administration of one or more agents that results in decreased TGF-β receptor activation is administered until the subject reaches at least 30 years of age. It starts when In some embodiments of any of the methods described herein, the first administration of one or more agents that results in decreased TGF-β receptor activation is administered until the subject reaches at least 40 years of age. It starts when In some embodiments of any of the methods described herein, the first administration of one or more agents that results in decreased TGF-β receptor activation is administered until the subject reaches at least 50 years of age. It starts when In some embodiments of any of the methods described herein, the first administration of one or more agents that results in decreased TGF-β receptor activation is administered until the subject reaches at least 60 years of age. It starts when

In some embodiments of any of the methods described herein, each of the two or more doses is about 0.01 mg/kg of each agent that results in decreased activation of the TGF-β receptor. ˜10 mg/kg of each agent that results in decreased activation of the TGF-β receptor. In some embodiments of any of the methods described herein, each of the two or more doses is about 0.02 mg/kg of each agent that results in decreased activation of the TGF-beta receptor. ˜5 mg/kg of each agent that results in decreased activation of the TGF-β receptor.

In some embodiments of any of the methods described herein, the subject has not been diagnosed or identified as having an age-related disease or an inflammatory disease. In some embodiments of any 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.

A method of treating an age-related disease or condition in a subject in need thereof, comprising administering to a subject identified as having an age-related disease or condition a therapeutically effective amount of one or more Provided herein are methods comprising administering a natural killer (NK) cell activator of

A method of killing senescent cells or reducing the number of senescent cells in a subject in need of killing senescent cells or reducing the number of senescent cells, comprising administering to the subject a therapeutically effective amount of one or more natural killers ( NK) Methods are also provided herein comprising administering a cell activator. In some embodiments of any of the methods described herein, the senescent cells are senescent cancer cells, senescent monocytes, senescent lymphocytes, senescent astrocytes, senescent microglia, senescent neurons, senescent tissue Fibroblasts, senescent skin fibroblasts, senescent keratinocytes, or other differentiated tissue-specific dividing functional cells. In some embodiments of any of the methods described herein, the senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced senescent cells. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an age-related disease or condition.

In some embodiments of any of the methods described herein, the age-related disease or condition is cancer, autoimmune disease, metabolic disease, neurodegenerative disease, cardiovascular disease, skin disease, It is selected from the group of progeria and frailty diseases. In some embodiments of any of the methods described herein, the cancer is solid tumor, hematologic tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyo sarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic Myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, 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, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

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

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

In some embodiments of any of the methods described herein, the neurodegenerative disease is selected from the group Alzheimer's disease, Parkinson's disease, and dementia.

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

In some embodiments of any of the methods described herein, the skin disorder is wound healing, alopecia, wrinkles, senile lentigines, skin thinning, xeroderma pigmentosum, and congenital Selected from the group of dyskeratosis.

In some embodiments of any of the methods described herein, the progeria is selected from the group of progeria and Hutchinson-Gilford progeria syndrome.

In some embodiments of any of the methods described herein, the fragility disease is selected from the group of fragility, response to vaccination, osteoporosis, and sarcopenia.

In some embodiments of any of the methods described herein, the age-related disease or condition is osteoarthritis, fat wasting, idiopathic pulmonary fibrosis, renal graft failure, liver fibrosis, bone mass loss, sarcopenia, age-related pulmonary tissue elastic loss, osteoporosis, age-related renal dysfunction, and chemically induced renal dysfunction.

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

In some embodiments of any of the methods described herein, administering results in a decrease in the number of senescent cells in the target tissue in the subject. In some embodiments of any of the methods described herein, adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature, bone tissue, central nervous system (CNS) tissue, ocular tissue, skin It is selected from the group of tissue, muscle tissue and secondary lymphoid organ tissue.

In some embodiments of any of the methods described herein, administering increases expression levels of CD25, CD69, mTORC1, SREBP1, IFN-γ, and granzyme B on activated NK cells bring.

A method of treating an age-related disease or condition in a subject in need of treatment of an age-related disease or condition, comprising administering to a subject identified as having an age-related disease or condition a therapeutically effective number of activated Also provided herein are methods comprising administering NK cells.

A method of killing senescent cells or reducing the number of senescent cells in a subject in need of killing senescent cells or reducing the number of senescent cells, comprising administering to the subject a therapeutically effective number of activated NK cells. Methods are also provided herein that include administering. In some embodiments of any of the methods described herein, the senescent cells are senescent cancer cells, senescent monocytes, senescent lymphocytes, senescent astrocytes, senescent microglia, senescent neurons, senescent tissue Fibroblasts, senescent skin fibroblasts, senescent keratinocytes, or other differentiated tissue-specific dividing functional cells. In some embodiments of any of the methods described herein, the senescent cancer cells are chemotherapy-induced senescent cells or radiation-induced senescent cells. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an age-related disease or condition.

In some embodiments of any of the methods described herein, the age-related disease or condition is cancer, autoimmune disease, metabolic disease, neurodegenerative disease, cardiovascular disease, skin disease, It is selected from the group of progeria and frailty diseases. In some embodiments of any of the methods described herein, the cancer is solid tumor, hematologic tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyo sarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic Myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, 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, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

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

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

In some embodiments of any of the methods described herein, the neurodegenerative disease is selected from the group Alzheimer's disease, Parkinson's disease, and dementia.

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

In some embodiments of any of the methods described herein, the skin disorder is wound healing, alopecia, wrinkles, senile lentigines, skin thinning, xeroderma pigmentosum, and congenital Selected from the group of dyskeratosis.

In some embodiments of any of the methods described herein, the progeria is selected from the group of progeria and Hutchinson-Gilford progeria syndrome.

In some embodiments of any of the methods described herein, the fragility disease is selected from the group of fragility, response to vaccination, osteoporosis, and sarcopenia.

In some embodiments of any of the methods described herein, the age-related disease or condition is age-related macular degeneration, osteoarthritis, fat wasting, idiopathic pulmonary fibrosis, renal transplant failure, selected from the group of liver fibrosis, bone loss, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis, age-related renal dysfunction, and chemically induced renal dysfunction.

Some embodiments of any of the methods described herein involve obtaining resting NK cells and resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators. contacting with, which contacting results in the generation of activated NK cells that are then administered to the subject. In some embodiments of any of the methods described herein, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of any of the methods described herein, the resting NK cells are allogeneic resting NK cells. In some embodiments of any of the methods described herein, the resting NK cells are induced NK cells. In some embodiments of any of the methods described herein, the resting NK cells are haploidentical resting NK cells. In some embodiments of any of the methods described herein, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors. Some embodiments of any of the methods described herein further comprise isolating the activated NK cells before the activated NK cells are administered to the subject. Some embodiments of any of the methods described herein involve administering a nucleic acid encoding a chimeric antigen receptor or recombinant T cell receptor to resting NK cells or activated NK cells prior to administration to a subject. Further comprising introducing into NK cells.

A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time comprising administering to the subject a therapeutically effective amount of 1 Also provided herein are methods comprising administering one or more natural killer (NK) cell activators.

1. A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time, comprising administering to said subject a therapeutically effective number of active Also provided herein are methods comprising administering the transformed NK cells. Some embodiments of any of the methods described herein involve obtaining resting NK cells and resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators. contacting with, which contacting results in the generation of activated NK cells that are then administered to the subject. In some embodiments of any of the methods described herein, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of any of the methods described herein, the resting NK cells are allogeneic resting NK cells. In some embodiments of any of the methods described herein, the resting NK cells are induced NK cells. In some embodiments of any of the methods described herein, the resting NK cells are haploidentical resting NK cells. In some embodiments of any of the methods described herein, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors. Some embodiments of any of the methods described herein further comprise isolating the activated NK cells before the activated NK cells are administered to the subject.

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

In some embodiments of any of the methods described herein, the time period is from about 1 month to about 10 years. In some embodiments of any of the methods described herein, the method results in a decrease in the number of senescent skin fibroblasts in the subject's skin over time.

A method of helping treat obesity in a subject in need of help treating obesity over a period of time comprising administering to the subject a therapeutically effective amount of one or more natural killer (NK) cell activators. A method is also provided herein, comprising:

Also herein is a method of helping treat obesity in a subject in need of help treating obesity over a period of time comprising administering to the subject a therapeutically effective number of activated NK cells. provided in the book. Some embodiments of any of the methods described herein involve obtaining resting NK cells and resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators. contacting with, which contacting results in the generation of activated NK cells that are then administered to the subject. In some embodiments of any of the methods described herein, the resting NK cells are autologous NK cells obtained from the subject. In some embodiments of any of the methods described herein, the resting NK cells are allogeneic resting NK cells. In some embodiments of any of the methods described herein, the resting NK cells are induced NK cells. In some embodiments of any of the methods described herein, the resting NK cells are haploidentical resting NK cells. In some embodiments of any of the methods described herein, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors. Some embodiments of any of the methods described herein further comprise isolating the activated NK cells before the activated NK cells are administered to the subject.

In some embodiments of any of the methods described herein, the method results in weight loss in the subject over a period of time. In some embodiments of any of the methods described herein, the method results in a decrease in body mass index (BMI) of the subject over time. In some embodiments of any of the methods described herein, the method results in a reduction in the rate of progression from pre-diabetes to type 2 diabetes in the subject. In some embodiments of any of the methods described herein, the method results in decreased fasting blood glucose levels in the subject. In some embodiments of any of the methods described herein, the method results in increased insulin sensitivity in the subject. In some embodiments of any of the methods described herein, the method results in a reduction in the severity of atherosclerosis in the subject. In some embodiments of any of the methods described herein, the time period is from about 2 weeks to about 10 years.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators is 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, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, and KIR3DS1.

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

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

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

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

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

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

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

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in activation of CD25 or CD59 receptors is CD25 or CD59 is an agonistic antibody that specifically binds to

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

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

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

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

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

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

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in NKG2D receptor activation is specifically directed to NKG2D It is an agonistic antibody that binds.

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in activation of the KIR2DS2/3 receptor is KIR2DS2/3 is an agonistic antibody that specifically binds to

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in activation of the KIR2DL4 receptor is specifically It is an agonistic antibody that binds.

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

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

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators is PD-1, TGF-beta receptor, TIGIT, CD1 , TIM-3, Siglec-7, IRP60, Tactile, IL1R8, NKG2A/KLRD1, KIR2DL1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2 result in a decrease in In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of PD-1 is PD-1 soluble 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 result in decreased TGF-β receptor activation is a soluble A TGF-beta receptor, an antibody that specifically binds to TGF-beta, or an antagonistic antibody that specifically binds to a TGF-beta receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of TIGIT is specifically An antagonist antibody that binds, a soluble TIGIT, or an antibody that specifically binds to a ligand of TIGIT.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of CD1 is specifically An antagonist antibody that binds, soluble CD1, or an antibody that specifically binds to a ligand of CD1.

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

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of IRP60 is specifically An antagonist antibody that binds, or an antibody that specifically binds to a ligand of IRP60.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of Tactile is specifically An antagonist antibody that binds, or an antibody that specifically binds to a ligand of Tactile.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of IL1R8 is specifically An antagonist antibody that binds, or an antibody that specifically binds to a ligand of IL1R8.

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of KIR2DL1 is specifically An antagonist antibody that binds, or an antibody that specifically binds to a ligand of KIR2DL1.

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of KIR2DL5 is specifically An antagonist antibody that binds, or an antibody that specifically binds to a ligand of KIR2DL5.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of KIR3DL1 is specifically An antagonist antibody that binds, or an antibody that specifically binds to a ligand of KIR3DL1.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of KIR3DL2 is specifically An antagonist antibody that binds, or an antibody that specifically binds to a ligand of KIR3DL2.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators that result in decreased activation of ILT2/LIR-1 is ILT2 An antagonist antibody that specifically binds to /LIR-1 or an antibody that specifically binds to a ligand of ILT2/LIR-1.

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

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators comprises (i) the first target binding domain, (ii) A single chain chimeric polypeptide comprising 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 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 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 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 are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises a scFv or 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 are CD16a, CD33, CD20, CD19, CD22, CD123 , PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2 , CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD , TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell binding to a target selected from the group of factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor do.

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 are soluble interleukin or cytokine proteins. In some embodiments of any of the methods described herein, the soluble interleukin or cytokine protein is 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-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor soluble IL-4 receptor, or soluble IL-10 receptor.

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 that is 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 that is 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 that is at least 95% identical to SEQ ID NO:93. In some embodiments of any of the methods described herein, the soluble human tissue factor domain has a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein. Isoleucine at the amino acid position corresponding to amino acid position 22 of the human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, and amino acid position 58 of the mature wild-type human tissue factor protein. tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; It does not contain one or more of the phenylalanines at the amino acid position corresponding to amino acid position 140 of the wild-type human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble human tissue factor domain has a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein. Isoleucine at the amino acid position corresponding to amino acid position 22 of the human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, and amino acid position 58 of the mature wild-type human tissue factor protein. tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; None of the phenylalanine is present at the amino acid position corresponding to amino acid position 140 of the wild-type human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is incapable of binding Factor VIIa. 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 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 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 are a first target binding domain, a second target binding domain, or a soluble tissue factor domain. directly adjacent to In some embodiments of any of the methods described herein, the single chain chimeric polypeptide comprises one of the at least one additional target binding domain and the first target binding domain, the second 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 C-terminus. In some embodiments of any of the methods described herein, one of the one or more additional target binding domains is the first target binding domain, the second target binding domain, or directly adjacent to the soluble tissue factor domain. In some embodiments of any of the methods described herein, the single chain chimeric polypeptide comprises one of the at least one additional target binding domain and the first target binding domain, the second 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 of the one or more additional antigen binding domains at the N-terminus is the first target binding domain, the second target Directly adjacent to the binding domain, or soluble tissue factor domain. In some embodiments of any of the methods described herein, the single chain chimeric polypeptide comprises one of the one or more additional antigen binding domains at the N-terminus and the first target binding domain. Further comprising a linker sequence between the 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 at the C-terminus is the first target binding domain, the second target Directly adjacent to the binding domain, or soluble tissue factor domain. In some embodiments of any of the methods described herein, the single chain chimeric polypeptide comprises one of the one or more additional antigen binding domains at the C-terminus and the first target binding domain. Further comprising a linker sequence between the 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 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 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 one or more additional target binding domains contain 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 bind to the same antigen. Binds specifically. 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 directed to the same epitope. Binds specifically. 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 have the same amino acid sequence including.

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 specific for different antigens. physically connect.

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 It is an antigen-binding domain. 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 an antigen binding domain is. In some embodiments of any of the methods described herein, the antigen binding domain comprises a scFv or 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 are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand , scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 It specifically binds to a target selected from the group of receptors.

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 one or more additional target binding domains is a soluble interleukin or cytokine protein. In some embodiments of any of the methods described herein, the soluble interleukin or cytokine protein is 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 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-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL -4 receptor, or soluble IL-10 receptor.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators comprises (a)(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) (i) a second domain of the pair of affinity domains and (ii) a second chimeric polypeptide comprising a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide comprise a pair of affinity domains associates through binding of the first and second domains of

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 within the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide has a further includes a linker sequence. In some embodiments of any of the methods described herein, the soluble tissue factor domain and the first domain of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. there is In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a first affinity domain paired with a soluble tissue factor domain within the first chimeric polypeptide. It further contains a linker sequence between the domains. In some embodiments of any of the methods described herein, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. are doing. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises the second domain and the second of the pair of affinity domains within the second chimeric polypeptide. It further comprises a linker sequence between it and the 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 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 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 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 are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises a scFv or 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 are CD16a, CD33, CD20, CD19, CD22, CD123 , PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2 , CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD , TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell specific for a target selected from the group of factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor physically connect.

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 are soluble interleukin or cytokine proteins. In some embodiments of any of the methods described herein, the soluble interleukin or cytokine protein is 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-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL -4 receptor, or soluble IL-10 receptor.

In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains and one or more additional antigen binding domains at least one of which is positioned between the soluble tissue factor domain and the first of the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide is 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 of the pair of affinity domains.

In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises one or more additional targets at the N-terminus and/or C-terminus of the first chimeric polypeptide. Further comprising a 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 a pair of affinity domains within the first chimeric polypeptide. directly adjacent to the first domain of In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises at least one of the one or more additional target binding domains and a first pair of affinity domains. It further contains a linker sequence between one domain. In some embodiments of any of the methods described herein, at least one of the one or more additional target binding domains is the first target binding domain within the first chimeric polypeptide. Directly adjacent to the domain. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises at least one of the one or more additional target binding domains and the first target binding domain. further includes a linker sequence between

In some embodiments of any of the methods described herein, at least one of the one or more additional target binding domains are N-terminal and/or C-terminal of the first chimeric polypeptide. terminally positioned, at least one of the one or more additional target binding domains is between the soluble tissue factor domain and the first of the pair of affinity domains in the first chimeric polypeptide is positioned in In some embodiments of any of the methods described herein, at least one additional target binding domain of the one or more additional target binding domains located N-terminally comprises the first immediately adjacent to the first target binding domain or the first domain of a pair of affinity domains within the chimeric polypeptide of . In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain. Further comprising a linker sequence interposed between the domain or the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, at least one additional target binding domain of the one or more additional target binding domains located C-terminally comprises the first immediately adjacent to the target binding domain or the first domain of the first pair of affinity domains in the chimeric polypeptide of . In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain. Further comprising a linker sequence interposed between the domain or the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains At least one of the domains is directly adjacent to 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 comprises (i) a soluble tissue factor domain and a first affinity domain paired with the soluble tissue factor domain; and/or (ii) the first domain of the pair of affinity domains and the soluble tissue factor domain; Further comprising a linker sequence disposed between at least one of the one or more additional target binding domains positioned between the first domain of the pair of affinity domains.

In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises one or more additional targets at the N-terminus and/or C-terminus of the second chimeric polypeptide. Further comprising a 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 a pair of affinity domains within the second chimeric polypeptide. directly adjacent to the second domain of In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises at least one of the one or more additional target binding domains within the second chimeric polypeptide. Further comprising a linker sequence between the second domain of 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 is a second target binding domain within the second chimeric polypeptide. Directly adjacent to the domain. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises at least one of the one or more additional target binding domains within the second chimeric polypeptide. further comprising a linker sequence between the domain 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 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 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 one or more additional target binding domains contain 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 bind to the same antigen. Binds specifically. 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 directed to the same epitope. Binds specifically. 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 have the same amino acid sequence including.

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 specific for different antigens. physically connect. 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 It is an antigen-binding domain. 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 an antigen binding domain is. In some embodiments of any of the methods described herein, the antigen binding domain comprises 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 are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand , scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 It specifically binds to a target selected from the group of receptors.

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 one or more additional target binding domains is a soluble interleukin or cytokine protein. In some embodiments of any of the methods described herein, the soluble interleukin or cytokine protein is 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 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-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL -4 receptor, or soluble IL-10 receptor.

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 that is 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 that is 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 that is at least 95% identical to SEQ ID NO:93.

In some embodiments of any of the methods described herein, the soluble human tissue factor domain has a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein. Isoleucine at the amino acid position corresponding to amino acid position 22 of the human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, and amino acid position 58 of the mature wild-type human tissue factor protein. tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; It does not contain one or more of the phenylalanines at the amino acid position corresponding to amino acid position 140 of the wild-type human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble human tissue factor domain has a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein. Isoleucine at the amino acid position corresponding to amino acid position 22 of the human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, and amino acid position 58 of the mature wild-type human tissue factor protein. tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; None of the phenylalanine is present at the amino acid position corresponding to amino acid position 140 of the wild-type human tissue factor protein.

In some embodiments of any of the methods described herein, the soluble tissue factor domain is unable to bind Factor VIIa. 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 of the methods described herein, the multichain 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 a sushi domain derived from the alpha chain of the human IL-15 receptor (IL-15Rα) and a soluble IL-15Rα 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 human IL-15Rα is mature full-length IL-15Rα.

In some embodiments of any of the methods described herein, the pair of affinity domains are barnase and barnstar, PKA and AKAP, an adapter/docking tag module based on a mutated RNase I fragment, and It is selected from the group of SNARE modules based on the interaction of proteins syntaxin, synaptotagmin, synaptobrevin and SNAP25.

In some embodiments of any of the methods described herein, at least one of the one or more NK cell activators is (a) the first and second chimeric polypeptides first and second chimeric polypeptides, each comprising (i) a first target binding domain, (ii) an Fc domain, and (iii) a first of a pair of affinity domains; b) third and fourth chimeric polypeptides, each comprising (i) a second of a pair of affinity domains and (ii) a second target binding domain; a chimeric polypeptide, wherein the first and second chimeric polypeptides and the third and fourth chimeric polypeptides are a first domain and a second domain of a pair of affinity domains; and the first and second chimeric polypeptides associate through their Fc domains.

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

In some embodiments of any of the methods described herein, the second domain of the pair of affinity domains and the second target binding domain are within the third and fourth chimeric polypeptides directly adjacent to each other. In some embodiments of any of the methods described herein, the third and fourth chimeric polypeptides are the second of the pair of affinity domains within 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 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 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 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 are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises a scFv or 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 are CD16a, CD33, CD20, CD19, CD22, CD123 , PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2 , CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD , TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell specific for a target selected from the group of factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor physically connect.

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 are soluble interleukin or cytokine proteins. In some embodiments of any of the methods described herein, the soluble interleukin or cytokine protein is 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-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL -4 receptor, or soluble IL-10 receptor.

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 from wild-type soluble human tissue factor.

Killing spontaneous and/or treatment-induced senescent cells or reducing the number of 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 is a method of causing.

Also herein are methods of reducing spontaneous and/or treatment-induced accumulation of 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.

Also herein are methods of reducing the levels of markers of spontaneous 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. provided in the book.

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

Levels of one or more SASP factors derived from spontaneous 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 activity and/or activity.

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

In some embodiments, the age-related disease is Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, disc degeneration, osteoarthritis , diabetes mellitus type 2, lipodystrophy, lipodystrophy, atherosclerosis, cataract, COPD, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, bone Arthritis, Parkinson's disease, age-related pulmonary tissue elastic loss, age-related macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia disease, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

In some embodiments, the age-related disease is solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B cell Neoplasm, multiple myeloma, B cell lymphoma, B cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia ( ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer , colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma It is.

In some embodiments, the inflammatory disease is 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-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders, and cancer therapy-related cognitive impairment.

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 spontaneous and/or treatment-induced senescent cells in target tissues in a subject. In some embodiments, the target tissue is adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and Selected from the group of secondary lymphoid organ tissues.

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

In some embodiments, at least one of the one or more common gamma chain family cytokine receptor activators comprises (i) the first target binding domain, (ii) the soluble tissue factor domain, and (iii) ) a single chain chimeric polypeptide comprising a second target binding domain, wherein one or both of the first target binding domain and the second target binding domain are a soluble common gamma chain family cytokine, a common gamma chain family An agonist antigen binding domain that specifically binds to a 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 comprise 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 be done. In some embodiments, one or both of the first target binding domain and the second target binding domain comprise an agonist 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. be. In some embodiments, the agonist 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 the same antigen. In some embodiments, the first target binding domain and the second target binding domain specifically bind 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 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 that is 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 comprises (a) (i) a first target binding domain, (ii) a soluble tissue factor domain; and (iii) a first chimeric polypeptide comprising a first of a pair of affinity domains, and (b) (i) a second of the pair of affinity domains, and (ii) a second target binding a second chimeric polypeptide comprising a domain, wherein one or both of the first target binding domain and the second target binding domain are a soluble common gamma chain family cytokine, the common An agonist antigen binding domain that specifically binds a gamma chain family cytokine receptor, a soluble common gamma chain family cytokine receptor, or an antigen binding domain that specifically binds a common gamma chain family cytokine.

In some embodiments, one or both of the first target binding domain and the second target binding domain comprise a soluble common gamma chain family cytokine. In some embodiments, the soluble common gamma chain family cytokine is selected from the group of soluble IL-2, soluble IL-4, soluble IL-7, soluble IL-9, soluble IL-15, and soluble IL-21. be. In some embodiments, one or both of the first target binding domain and the second target binding domain comprise an agonist 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. be. In some embodiments, the agonist 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 within 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 within 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 within the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. In some embodiments, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide.

In some embodiments, the first target binding domain and the second target binding domain specifically bind the same antigen. In some embodiments, the first target binding domain and the second target binding domain specifically bind 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 that is 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 (IL15Rα) and soluble IL-15. In some embodiments, the paired affinity domains are barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 interactions. It is selected from a group of SNARE modules based on

In some embodiments, the first domain or the second domain of the pair of affinity domains is an antigen binding domain that specifically binds to a soluble common gamma chain family cytokine or 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 soluble IL-15, or an IL-15 agonist. In some embodiments, the soluble IL-15 is at least 90% identical to SEQ ID NO:82. In some embodiments, the IL-15 agonist comprises a complex of IL-15 and all or part of the soluble IL-15 receptor (IL-15R). In some embodiments, the portion of soluble IL-15R is a portion of IL-15Rα. In some embodiments, the portion of 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, the 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 to a common gamma chain family cytokine.

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

In some embodiments, one, two or more doses are administered by subcutaneous administration. In some embodiments, two or more doses are administered by intramuscular administration. 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 from about 0.01 mg/kg of each common gamma chain family cytokine receptor activator to about 10 mg/kg of each common gamma chain family cytokine receptor activator. Dosage is administered. In some embodiments, each of the two or more doses is from about 0.02 mg/kg of each common gamma chain family cytokine receptor activator to about 5 mg/kg of each common gamma chain family cytokine receptor activator. Dosage is administered.

In some embodiments, the first administration 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 administration 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 administration 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 administration 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 has not been diagnosed or identified as having an age-related disease or an 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 decreased activation of TGF-β receptors. In some embodiments, the agent that results in decreased TGF-beta receptor activation is a soluble TGF-beta receptor, an extracellular domain of a TGF-beta receptor, an antibody that specifically binds to TGF-beta , an antagonist antibody that binds to the TGF-β receptor, an agent that binds to LAP, or an agent that binds to the TGF-β/LAP complex. In some embodiments, the one or more agents that result in decreased activation of TGF-β are activated TGF-β receptors through binding to LAP or to the TGF-β/LAP complex. decrease.

In some embodiments, the soluble human tissue factor domain does not initiate blood clotting. In some embodiments, the method further comprises administering an additional therapeutic agent selected from the group 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° C. for at least 10 days. In some embodiments of any of the methods described herein, the multichain chimeric polypeptide is stable in human serum at 37° C. 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 multichain chimeric polypeptide does not have significant clotting activity.

In some embodiments of any of the methods described herein, the method results in rejuvenation of aged immune cells in the subject. In some embodiments of any of the methods described herein, rejuvenation of aged 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 senescent immune cells include senescent NK cells, senescent NKT cells, senescent T cells, senescent B cells, senescent single cells. One or more of spheres, senescent macrophages, senescent neutrophils, senescent basophils, senescent eosinophils, senescent Kupffer cells, and senescent microgial cells. In some embodiments of any of the methods described herein, diseased cells include cancer cells, virus-infected cells, and intracellular bacteria-infected cells. In some embodiments of any of the methods described herein, infectious agents include viruses, bacteria, fungi, and parasites.

As used herein, the term "chimera" refers to amino acid sequences (e.g., two different naturally occurring proteins, originally from two different sources, e.g., from the same or different species) ( For example, it refers to a polypeptide comprising a domain). For example, a chimeric polypeptide can contain domains from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can include domains that are synthetic sequences (eg, scFv) and domains derived from naturally occurring proteins (eg, naturally occurring human proteins). In some embodiments, a chimeric polypeptide can comprise at least two different domains (eg, two different scFvs) that are synthetic sequences.

An "activated NK cell" is, for example, two or more of CD25, CD69, MTOR-C1, SREBP, IFN-γ, and a granzyme (e.g., granzyme B) compared to a resting NK cell (e.g., , 3, 4, 5, or 6) NK cells showing increased expression levels. Exemplary methods for identifying expression levels of CD25, CD69, MTOR-C1, SREBP, IFN-γ, and granzymes (eg, granzyme B) are described herein.

A “resting NK cell” is, for example, two or more of CD25, CD69, MTOR-C1, SREBP, IFN-γ, and a granzyme (eg, granzyme B) compared to an activated NK cell (eg, , 3, 4, 5, or 6) NK cells with decreased expression.

An "NK cell activator" is an agent that induces or promotes (alone or in combination with additional NK cell activators) resting NK cells to evolve 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 (e.g., formed from amino acids from a single polypeptide, or formed from two or more antigens) capable of specifically binding to one or more different antigens. (eg, formed from amino acids from the same or different polypeptides). In some instances, an antigen binding domain can bind an antigen or epitope with specificity and affinity similar to a naturally occurring antibody. In some embodiments, the antigen binding domain can be an antibody or fragment thereof. In some embodiments, antigen binding domains may include alternative scaffolds. Non-limiting examples of antigen binding domains are described herein. Additional examples of antigen binding domains are known in the art.

A "soluble tissue factor domain" is at least 70% identical (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) Point. Non-limiting examples of soluble tissue factor domains are described herein.

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

The term "soluble interleukin receptor" is used in the broadest sense herein and refers to one of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). Refers to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) capable of binding more than one. For example, the soluble interleukin receptor 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) and retains the ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (optionally also the intracellular domain). ) sequence. Non-limiting examples of soluble interleukin receptors are described herein.

The term "soluble cytokine receptor" is used in the broadest sense herein and refers to one of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). It refers to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) capable of binding to a molecule. For example, the soluble cytokine receptor 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) and retains the ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (optionally also lacking an intracellular domain). ) may contain an array. 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 contain one or more antigen binding domains that specifically bind an antigen or epitope. Antibodies specifically include, for example, intact antibodies (eg, intact immunoglobulins), antibody fragments, and multispecific antibodies. One example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

"Affinity" refers to the total strength of non-covalent interactions between an antigen-binding site and its binding partner (eg, antigen or epitope). Unless otherwise indicated, "affinity" as used herein refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of an antigen binding domain and an antigen or epitope. The affinity of molecule X for its partner Y can be expressed as a dissociation equilibrium constant (K _D ). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (eg, BIACORE®) or biolayer interferometry (eg, FORTEBIO®). Additional methods for determining affinity for antigen binding domains and their corresponding antigens or epitopes 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 two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) protein chains (e.g., at least a first chimeric It refers to a polypeptide, including a polypeptide and a second polypeptide), in which two or more protein chains are joined via non-covalent bonds to form a quaternary structure.

The term "affinity domain pair" means less than 1×10 ⁻⁷ M (eg, less than 1×10 ⁻⁸ M, less than 1×10 ⁻⁹ M, less than 1×10 ⁻¹⁰ M, or less than 1×10 ⁻¹¹ M) are two different protein domains that specifically bind to each other with a K _D less than M). In some examples, the pair of affinity domains can be a pair of naturally occurring proteins. In some embodiments, the pair of affinity domains can be a pair of synthetic proteins. Non-limiting examples of paired affinity domains are described herein.

The term "epitope" refers to that portion of an antigen that specifically binds to the antigen binding domain. Epitopes can consist, for example, of surface accessible amino acid residues and/or sugar side chains and can have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not the latter, can be lost in the presence of denaturing solvents. An epitope can 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 bound by antigen binding domains are known in the art.

The term "treating" means ameliorating at least one symptom of a disorder. In some examples, the disorder being treated is cancer and ameliorating at least one symptom of cancer is reducing abnormal proliferation, gene expression, signaling, translation, and/or secretion of factors. including. Generally, methods of treatment comprise administering to a subject in need of such treatment or determined to be in need of such treatment a therapeutically effective amount of a composition that reduces at least one symptom 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 are described herein for use in the present invention, and other suitable methods and materials known in the art can 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 become apparent from the following detailed description and figures, and from the claims.

Figures 1A-1B show the results of immunostimulation of exemplary multi-chain polypeptides in C57BL/6 mice. FIG. 1A shows spleen weights of mice treated with increasing doses of an exemplary multichain polypeptide compared to mice treated with control solution. FIG. 1B shows the percentage of immune cell types present in the spleen of mice treated with increasing doses of an exemplary multichain polypeptide compared to mice treated with control solution. Figures 2A-2B show the duration of immune stimulation of exemplary multi-chain polypeptides in C57BL/6 mice. FIG. 2A shows spleen weights over 92 hours in mice treated with 3 mg/kg of an exemplary multichain polypeptide. FIG. 2B shows the percentage of immune cell types present in the spleen over 92 hours in mice treated with 3 mg/kg of an exemplary multichain polypeptide. Figures 3A-3B show Ki67 and granzyme B expression in immune cells induced by exemplary multichain polypeptides. FIG. 3A shows Ki67 expression on CD4 ⁺ T cells, CD8 ⁺ T cells, natural killer (NK) cells, and CD19 ⁺ B cells at various time points after treatment with multichain polypeptides. FIG. 3B shows granzyme B expression in CD4 ⁺ T cells, CD8 ⁺ T cells, natural killer (NK) cells, and CD19 ⁺ B cells at various time points after treatment with multichain polypeptides. Shows the effect of tumor inhibition by splenocytes prepared from mice treated with exemplary multichain polypeptides at various time points after treatment. 5A-5B show control diet, high fat diet, untreated B6.129P2-ApoE ^tm1Unc /J mice (purchased from The Jackson Laboratory), and high fat diet, TGFRt15-TGFRs, 2t2, or 21t15. - Percentage and proliferation rate of CD4 ⁺ T cells, CD8 ⁺ T cells, natural killer (NK) cells, CD19 ⁺ B cells in the blood of mice treated with TGFRs. FIG. 5A shows the percentage of different cell types in each control and experimental group. FIG. 5B shows the growth rate of different cell types in each control and experimental group. Figures 6A-6E show exemplary physical appearances of mice fed either a control diet or a high-fat diet and either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs. Fasting body weights of untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs, fed either a control diet or a high-fat diet are shown. Fasting blood glucose levels of untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs, fed either a control diet or a high-fat diet are shown. Figures 9A-9F show chemotherapy-induced senescent B16F10 cells and senescent gene expression. FIG. 9A shows chemotherapy induction of senescent B16F10 cells visualized using SAβ-gal staining. Figures 9B-9F show the expression of p21 ^CIP1 , IL6, DPP4, RATE1E, and ULBP1 in chemotherapy-induced senescent B16F10 cells over time. Figures 10A-10F show colony formation and expression of stem cell markers by chemotherapy-induced senescent B16F10 cells. FIG. 10A shows colony formation by chemotherapy-induced senescent B16F10 cells. Figures 10B and 10C show Oct4 and Notch4 mRNA expression by chemotherapy-induced senescent B16F10 cells compared to control B16F10 cells. Figures 10D-10F show the percentage of double-positive chemotherapy-induced senescent B16F10 cells against two of three stem cell markers, including CD44, CD24, and CD133. FIGS. 11A-11C show migration and invasiveness of chemotherapy-induced senescent B16F10 cells. FIG. 11A shows the results of a migration assay comparing chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) to control B16F10 cells. Figures 11B and 11C show the results of an invasion assay comparing chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) to control B16F10 cells. Figures 12A and 12B show in vitro expanded NK cells and their cytotoxicity against chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) or control B16F10 cells. FIG. 12A shows an exemplary schematic of the process of obtaining in vitro expanded NK cells. FIG. 12B shows cytotoxicity of proliferated NK cells against chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) or control B16F10 cells. Figures 13A-13C show the results of combination therapy using a mouse melanoma model. FIG. 13A shows an exemplary schematic for treating melanoma in a mouse model. Figures 13B and 13C show changes in tumor volume over time with combination treatment comprising TGFRt15-TGFRs compared to chemotherapy or TA99 treatment alone. Shows the induction of senescence in the human pancreatic tumor cell line SW1990 and the expression of CD44 and CD24 in senescent SW1990 cells compared to control SW1990 cells. Expression of senescent markers by chemotherapy-induced senescent SW1990 cells. Cytotoxicity of in vitro activated human NK cells against chemotherapy-induced senescent SW1990 cells or control SW1990 cells. Schematic diagrams of exemplary IL-12/IL-15RαSu DNA constructs are shown. Schematic representation of an exemplary IL-18/TF/IL-15 DNA construct is shown. Schematic representation of interaction between exemplary IL-12/IL-15RαSu DNA constructs and IL-18/TF/IL-15 DNA constructs. Exemplary IL-12/IL-15RαSu fusion proteins and IL-18/TF/IL-15 fusions resulting in IL-18/TF/IL-15:IL-12/IL-15RαSu complex (18t15-12s) Schematic representation of interactions between proteins. A chromatograph of 18t15-12s purified elution from an anti-TF antibody affinity column is shown. Figure 3 shows an exemplary chromatographic profile of anti-TF antibody/SEC purified 18t15-12s protein after elution on an analytical size exclusion column, showing separation of monomeric multiprotein 18t15-12s complexes from protein aggregates. . An example of 4-12% SDS-PAGE of 18t15-12s complex after reduction of disulfide bonds is shown. Lane 1: SeeBlue Plus2 marker, lane 2: anti-TF Ab purified 18t15-12s (0.5 μg), lane 3: anti-TF Ab purified 18t15-12s (1 μg). SDS PAGE analysis of deglycosylated and non-deglycosylated 18t15-12s is shown. Lane 1: anti-TF Ab purified 18t15-12s (0.5 μg) (non-deglycosylated), lane 2: anti-TF Ab purified 18t15-12s (1 μg) (non-deglycosylated), lane 3: 18t15-12s (1 μg). ) (deglycosylation), lane 4: Mark12 unstained marker. Sandwich ELISA of 18t15-12s conjugate with anti-human tissue factor capture antibody and biotinylated anti-human IL-12 detection antibody (BAF219). Sandwich ELISA of 18t15-12s complex with anti-human tissue factor capture antibody and biotinylated anti-human IL-15 detection antibody (BAM247). Sandwich ELISA of 18t15-12s complex with anti-human tissue factor capture antibody and biotinylated anti-human IL-18 detection antibody (D045-6). Sandwich ELISA of 18t15-12s conjugates with anti-human tissue factor (I43) capture antibody and anti-human tissue factor detection antibody. Proliferation of IL-15-dependent 32Dβ cells mediated by 18t15-12s complex (open squares) and recombinant IL-15 (filled squares) is shown. Biological activity of IL-18 within the 18t15-12s complex (open squares) is shown, with recombinant IL-18 (filled squares) and recombinant IL-12 (filled circles) as positive and negative controls, respectively. Function. Biological activity of IL-12 within the 18t15-12s complex (open squares) is shown, with recombinant IL-12 (closed circles) and recombinant IL-18 (closed squares) as positive and negative controls, respectively. Function. Figures 32A and 32B show cell surface CD25 expression of NK cells induced by the 18t15-12s complex and cell surface CD69 expression of NK cells induced by the 18t15-12s complex. Flow cytometry graph of intracellular IFN-γ expression of NK cells induced by 18t15-12s complex. 18t15-12s-induced human NK cell cytotoxicity against K562 cells. Schematic representation of an exemplary IL-12/IL-15RαSu/αCD16 DNA construct is shown. Schematic representation of an exemplary IL-18/TF/IL-15 DNA construct is shown. Schematic representation of interaction between exemplary IL-12/IL-15RαSu/αCD16scFv DNA constructs and IL-18/TF/IL-15 DNA constructs. A schematic representation of an exemplary 18t15-12s/αCD16 protein complex is shown. Sandwich ELISA of 18t15-12s16 complexes containing anti-human tissue factor capture antibody and biotinylated anti-human IL-12 (BAF219) (dark line) or anti-human tissue factor detection antibody (light line). Schematic representation of an exemplary TGFβRII/IL-15RαSu DNA construct is shown. Schematic diagrams of exemplary IL-21/TF/IL-15 constructs are shown. Schematic representation of interaction between exemplary IL-IL-21/TF/IL-15 constructs and TGFβRII/IL-15RαSu constructs. Between exemplary TGFβRII/IL-15RαSu and IL-21/TF/IL-15 fusion proteins resulting in IL-21/TF/IL-15/TGFβRII/IL-15RαSu complexes (21t15-TGFRs) shows a schematic diagram of the interaction of A chromatograph of 21t15-TGFRs purified elution from an anti-TF antibody affinity column is shown. An exemplary 21t15-TGFRs size exclusion chromatograph showing the major protein and high molecular weight peaks is shown. An example of 4-12% SDS-PAGE of 21t15-TGFRs complexes after reduction of disulfide bonds is shown. Lane 1: Mark12 unstained marker (the number on the left indicates the molecular weight in kDa), Lane 2: 21t15-TGFRs (0.5 μg), Lane 3: 21t15-TGFRs (1 μg), Lane 4: 21t15-TGFRs ( deglycosylated) (1 μg), MW expected size 53 kDa and 39.08 kDa. Sandwich ELISA of 21t15-TGFRs conjugates with anti-human tissue factor capture antibody and biotinylated anti-human IL-21 detection antibody (13-7218-81, BioLegend). Sandwich ELISA of 21t15-TGFRs conjugates with anti-human tissue factor capture antibody and biotinylated anti-human IL-15 detection antibody (BAM247, R&D Systems). Sandwich ELISA of 21t15-TGFRs conjugates with anti-human tissue factor capture antibody and biotinylated anti-human TGFβRII detection antibody (BAF241, R&D Systems). Sandwich ELISA of 21t15-TGFRs conjugates with anti-human tissue factor (I43) capture antibody and anti-human tissue factor detection antibody. IL-15-dependent proliferation of 32Dβ cells mediated by the 21t15-TGFRs complex (open squares) compared to IL-15 (filled squares). Biological activity of the TGFβRII domain within the 21t15-TGFRs complex (open squares). TGFβRII/Fc (filled squares) served as a positive control. Flow cytometry graphs of cell surface CD25 expression of NK cells induced by 21t15-TGFRs complexes. Flow cytometry graph of cell surface CD69 expression of NK cells induced by 21t15-TGFRs complexes. Flow cytometry graph of intracellular IFN-γ expression of NK cells induced by 21t15-TGFRs complexes. 21t15-TGFRs-induced human NK cell cytotoxicity against K562 cells. Schematic representation of an exemplary αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide. FIG. 4 is a chromatograph showing elution of an exemplary αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide from an anti-tissue factor affinity column. FIG. 4 is a chromatograph showing elution of a Superdex 200 Increase 10/300 GL gel filtration column loaded with an exemplary αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide. 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. 2 is a graph showing ELISA quantitation of an exemplary αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide performed using the method described in Example 1. FIG. Purified tissue factor was used as a control. FIG. 4 is a graph showing the ability of an exemplary αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide to stimulate CD25 expression in CD4 ⁺ T cells isolated from blood from two donors. Experiments were performed as described in Example 2. FIG. 4 is a graph showing the ability of an exemplary αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide to stimulate CD25 expression in CD8 ⁺ T cells isolated from blood from two donors. Experiments were performed as described in Example 2. FIG. 2 is a graph showing the ability of an exemplary αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide to stimulate CD69 expression in CD4 ⁺ T cells isolated from blood from two donors. Experiments were performed as described in Example 2. Schematic representation of an exemplary IL-7/IL-15RαSu DNA construct is shown. Schematic representation of an exemplary IL-21/TF/IL-15 DNA construct is shown. Schematic representation of interaction between exemplary IL-7/IL-15RαSu DNA constructs and IL-21/TF/IL-15 DNA constructs. Exemplary IL-7/IL-15RαSu fusion protein and IL-21/TF/IL-15 fusion resulting in IL-21/TF/IL-15:IL-7/IL-15RαSu complex (21t15-7s) Schematic representation of interactions between proteins. Schematic diagrams of exemplary IL-21/IL-15RαSu DNA constructs are shown. Schematic representation of an exemplary IL-7/TF/IL-15 DNA construct is shown. Schematic representation of interaction between exemplary IL-21/IL-15RαSu DNA constructs and IL-7/TF/IL-15 DNA constructs. Exemplary IL-21/IL-15RαSu fusion protein and IL-7/TF/IL-15 fusion resulting in IL-7/TF/IL-15:IL-21/IL-15RαSu complex (7t15-21s) Schematic representation of interactions between proteins. Oxygen consumption rate (OCR) in picomoles/min of human NK cells (2×10 ⁶ cells/mL) isolated from the blood of two different donors. Figure 2 shows the extracellular acidification rate (ECAR) in mppH/min of human NK cells (2 x ¹⁰⁶ cells/mL) isolated from the blood of two different donors. A schematic representation of the 7t15-16s21 construct is shown. Additional schematics of the 7t15-16s21 construct are shown. Figures 77A and 77B show binding of 7t15-16s21 to CHO cells expressing human CD16b compared to control protein. Figures 78A-78C are the results of ELISA experiments using antibodies to IL-15, IL-21, and IL-7 to detect 7t15-16s21. Results of 32Dβ cell proliferation assays with 7t15-16s21 or recombinant IL-15 are shown. FIG. 2 shows the chromatographic profile of 7t15-16s21 protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin. Analytical SEC profile of 7t15-16s21 is shown. A schematic representation of the TGFRt15-16s21 construct is shown. Additional schematics of the TGFRt15-16s21 construct are shown. Figures 84A and 84B show the binding affinities of TGFRT15-16S21 and 7t15-21s to CHO cells expressing human CD16b. Figure 84A shows the binding affinity of TGFRT15-16S21 to CHO cells expressing human CD16b. FIG. 84B shows the binding affinity of 7t15-21s to CHO cells expressing human CD16b. Results of TGFβ1 inhibition by TGFRt15-16s21 and TGFR-Fc are shown. Results of 32Dβ cell proliferation assays using TGFRt15-16s21 or recombinant IL-15 are shown. Figures 87A-87C show the results of detection of IL-15, IL-21 and TGFβRII in TGFRt15-16s21 with the corresponding antibodies using ELISA. FIG. 2 shows the chromatographic profile of TGFRt15-16s21 protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin. Results of reducing SDS-PAGE analysis of TGFRt15-16s21 are shown. A schematic representation of the 7t15-7s construct is shown. Additional schematics of the 7t15-7s construct are shown. Chromatographic profile of 7t15-7s protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin is shown. Detection of TF, IL-15 and IL-7 in 7t15-7s using ELISA. Figures 94A and 94B show spleen weight and immune cell type percentages in 7t15-7s- and control-treated mice. FIG. 94A shows spleen weights in mice treated with 7t15-7s compared to PBS controls. FIG. 94B shows the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in mice treated with 7t15-7s compared to PBS controls. A schematic representation of the TGFRt15-TGFRs construct is shown. Additional schematics of TGFRt15-TGFRs constructs are shown. Results of TGFβ1 inhibition by TGFRt15-TGFRs and TGFR-Fc are shown. Results of 32Dβ cell proliferation assays using TGFRt15-TGFRs or recombinant IL-15 are shown. Figures 99A and 99B show the results of detection of IL-15 and TGFβRII in TGFRt15-TGFRs with the corresponding antibodies using ELISA. FIG. 4 is a line graph showing the chromatographic profile of TGFRt15-TGFRs protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin. Analytical SEC profiles of TGFRt15-TGFRs are shown. Pre-deglycosylation analyzed by reducing SDS-PAGE. TGFRt15-TGFRs after deglycosylation are shown. Figures 103A and 103B show spleen weight and immune cell type percentages in TGFRt15-TGFRs- and control-treated mice. FIG. 103A shows spleen weights in mice treated with TGFRt15-TGFRs compared to PBS controls. FIG. 103B shows the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in mice treated with TGFRt15-TGFRs compared to PBS controls. Figures 104A and 104B show spleen weight and immune stimulation over 92 hours in mice treated with TGFRt15-TGFRs. FIG. 104A shows spleen weights of mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. FIG. 104B shows the percentage of immune cells in mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figures 105A and 105B show Ki67 and granzyme B expression over time in mice treated with TGFRt15-TGFRs. FIG. 4 shows enhancement of splenocyte cytotoxicity by TGFRt15-TGFRs in C57BL/6 mice. Shown is the change in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or a combination of chemotherapy and TGFRt15-TGFRs in pancreatic cancer mouse models. Cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFRs. A schematic representation of the 7t15-21s137L (long version) construct is shown. Additional schematics of the 7t15-21s137L (long version) construct are shown. FIG. 4 is a line graph showing the chromatographic profile of 7t15-21s137L (long version) protein-containing cell culture supernatant after binding to and elution over anti-TF antibody resin. Analytical SEC profile of 7t15-21s137L (long version) is shown. Binding of 7t15-21s137L (short version) to CD137L (4.1BBL). Figures 114A-114C show detection of IL-15, IL21 and IL7 in 7t15-21s137L (short version) using ELISA. FIG. 114A shows detection of IL-15 in 7t15-21s137L (short version) using ELISA. FIG. 114B shows detection of IL21 in 7t15-21s137L (short version) using ELISA. FIG. 114C shows detection of IL7 in 7t15-21s137L (short version) using ELISA. Results of CTLL-2 cell proliferation assay are shown. Activity of 7t15-1s137L (short version) in promoting IL21R-containing B9 cell proliferation is shown. A schematic representation of the 7t15-TGFRs construct is shown. Additional schematics of 7t15-TGFRs constructs are shown. Results of TGFβ1 inhibition by 7t15-TGFRs and TGFR-Fc are shown. Figures 120A-120C show detection of IL-15, TGFβRII, and IL-7 in 7t15-TGFRs using ELISA. Results of 32Dβ cell proliferation assays using 7t15-TGFRs or recombinant IL-15 are shown. FIG. 4 is a line graph showing the chromatographic profile of 7t15-TGFRs protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin. 7t15-TGFRs before and after deglycosylation analyzed using reducing SDS-PAGE. ELISA detection of IL-7, IL-15 and TGFβRII in 7t15-TGFRs proteins is shown. Figures 125A and 125B show spleen weight and immune cell type percentages in 7t15-TGFRs- and control-treated mice. FIG. 125A shows spleen weights of mice treated with various dosages of 7t15-TGFRs compared to PBS controls. FIG. 125B shows the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in mice treated with various dosages of 7t15-TGFRs compared to PBS controls. Figures 126A and 126B show upregulation of CD44 expression on CD4 ⁺ and CD8 ⁺ T cells by 7t15-TGFRs in C57BL/6 mice. Figures 127A and 127B show upregulation of Ki67 and granzyme B expression on CD8 ⁺ T cells and NK cells by 7t15-TGFRs in C57BL/6 mice. FIG. 4 shows enhancement of splenocyte cytotoxicity by 7t15-TGFRs in C57BL/6 mice. A schematic representation of the TGFRt15-21s137L construct is shown. Additional schematics of the TGFRt15-21s137L construct are shown. FIG. 3 is a line graph showing the chromatographic profile of TGFRt15-21s137L protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin. A schematic representation of the TGFRt15-TGFRs21 construct is shown. Additional schematics of the TGFRt15-TGFRs21 construct are shown. FIG. 4 is a line graph showing the chromatographic profile of TGFRt15-TGFRs21 protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin. TGFRt15-TGFRs21 before and after deglycosylation analyzed by reducing SDS-PAGE. Figures 136A and 136B show detection of TGFRt15-TGFRs21 components using ELISA. Figures 137A and 137B show the percentage and proliferation of CD4 ⁺ T cells, CD8 ⁺ T cells, and natural killer (NK) cells present in the spleens of control-treated and TGFRt15-TGFRs21-treated mice. Shows upregulation of granzyme B expression in splenocytes in mice treated with TGFRt15-TGFRs21. FIG. 3 shows enhancement of splenocyte cytotoxicity by TGFRt15-TGFRs21 in C57BL/6 mice. A schematic representation of the TGFRt15-TGFRs16 construct is shown. Additional schematics of TGFRt15-TGFRs16 constructs are shown. A schematic representation of the TGFRt15-TGFRs137L construct is shown. Additional schematics of the TGFRt15-TGFRs137L construct are shown. Schematic diagram of an exemplary 2t2 single-chain chimeric polypeptide. IL-2 activity at 2t2 compared to recombinant IL-2 using the 32Dβ cell proliferation assay. IL-2 activity at 2t2 compared to recombinant IL-2 using CTLL-2 cell proliferation assay. Fasting blood glucose levels in ApoE ^−/− mice fed standard chow or high fat diet and treated with PBS control (untreated) or 2t2 are shown. Shown is the ratio of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cells in blood lymphocytes from ApoE ^−/− mice fed standard chow or high fat diet and treated with PBS control (untreated) or 2t2. FIG. 4 is a line graph showing the chromatographic profile of cell culture supernatant containing 2t2 protein after binding to and elution over anti-TF antibody resin. Analytical SEC profile of 2t2 is shown. Figures 151A and 151B show reducing SDS-PAGE analysis of 2t2 before and after deglycosylation. Figure 151A shows reducing SDS-PAGE analysis of 2t2 prior to deglycosylation. FIG. 151B shows reducing SDS-PAGE analysis of 2t2 after deglycosylation. Figures 152A and 152B show the results of immunostimulation of C57BL/6 mice with 2t2. FIG. 152A shows spleen weights after treatment with 2t2. Figure 152B shows the percentage of immune cell types after treatment with 2t2. Shows upregulation of CD25 expression of CD4 ⁺ T cells in mice treated with 2t2. 2 shows the pharmacokinetics of 2t2 in C57BL/6 mice. Figures 155A and 155B show the effect of 2t2 in attenuating high fat-induced atherosclerotic plaque formation in ApoE ^-/- mice. FIG. 155A shows representative images of atherosclerotic plaques from ApoE ^−/− mice fed standard chow or high fat diet and treated with either PBS control or 2t2. FIG. 155B shows the results of quantitative analysis of atherosclerotic plaques in each group. Fasting blood glucose levels in 2t2-treated mice compared to control-treated mice are shown. Shown is the percentage of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs in blood lymphocytes from 2t2- and control-treated mice. Schematic diagram of an exemplary 15t15 single-chain chimeric polypeptide. 15 shows IL-15 activity of 15t15 compared to recombinant IL-15 in the 32Dβ cell proliferation assay. FIG. 4 is a line graph showing the chromatographic profile of 15t15 protein-containing cell culture supernatant after binding to and elution over anti-TF antibody resin. Figures 161A and 161B show reducing SDS-PAGE analysis of 15t15 before and after deglycosylation. FIG. 161A shows reducing SDS-PAGE analysis of 15t15 prior to deglycosylation. FIG. 161B shows reducing SDS-PAGE analysis of 15t15 after deglycosylation. Figures 162A and 162B are a series of histograms (Figure 162A) and a series of graphs (Figure 162B) showing changes in the surface phenotype of NK cells after stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s plus anti-TF antibody. ). FIG. 3 is a series of graphs showing surface phenotype changes of lymphocyte populations following stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s. FIG. 4 is a series of graphs showing increased NK cell glycolysis after treatment with 18t15-12s. FIG. 4 is a series of graphs showing increased phospho-STAT4 and phospho-STAT5 levels in NK cells after stimulation with 18t15-12s. FIG. 4 is a series of graphs showing that overnight stimulation of NK cells with 18t15-12s enhances cell metabolism. Figures 167A-C are a series of graphs showing immune stimulation in C57BL/6 mice after treatment with 2t2. Figures 168A-B are a series of graphs showing immune stimulation in C57BL/6 mice after treatment with TGFRt15-TGFRs. Figures 169A-C are a series of graphs showing in vivo stimulation of Treg, NK and CD8 ⁺ T cells in ApoE ^−/− mice fed a Western diet and treated with TGFRt15-TGFRs or 2t2. Figures 170A-B are a series of graphs showing the induction of splenocyte proliferation by 2t2 in C57BL/6 mice. Figures 171A-C are a series of graphs showing immune stimulation in C57BL/6 mice after treatment with TGFRt15-TGFRs. Figures 172A-B are a series of graphs showing the in vivo induction of NK cell and CD8 ⁺ T cell proliferation in ApoE ^−/− mice fed a Western diet and treated with TGFRt15-TGFRs or 2t2. Schematic and series of graphs showing persistence of 7t15-21s and anti-TF antibody-proliferated NK cells in NSG mice after treatment with 7t15-21, TGFRt15-TGFRs, or 2t2. Figures 174A-B are a series of graphs showing enhanced NK cell cytotoxicity following treatment of NK cells with TGFRt15-TGFRs. Figures 175A-B are a series of graphs showing enhanced ADCC activity of NK cells following treatment of NK cells with TGFRt15-TGFRs. Figure 10 is a graph of in vitro killing of senescent B16F10 melanoma cells by TGFRt15-TGFRs/2t2 activated mouse NK cells. Figures 177A-H are a series of graphs showing anti-tumor activity of TGFRt15-TGFRs plus anti-TRP1 antibody (TA99) in combination with chemotherapy in a melanoma mouse model. Figures 178A-C are a series of graphs showing amelioration of western diet-induced hyperglycemia in ApoE ^−/− mice by 2t2. FIG. 4 is a series of graphs showing cell surface staining summarizing the differentiation of NK cells into cytokine-induced memory-like NK cells (CIML-NK cells) after stimulation with 18t15-12s and culture in rhIL-15. Shows upregulation of CD44 memory T cells. Top panel shows upregulation of CD44 memory T cells upon treatment with TGFRt15-TGFRs. Bottom panel shows upregulation of CD44 memory T cells upon treatment with 2t2. Figures 181A and 181B show improvement in hair growth after depilation in mice treated with 2t2 or IL-2. FIG. 181A shows skin pigmentation 10 days after depilation in mice treated with PBS, 2t2, or IL-2. FIG. 181B shows percent pigmentation of mice treated with PBS, 2t2, or IL-2, analyzed using ImageJ software. 14 shows skin pigmentation 14 days after depilation in mice treated with PBS, 2t2, IL-2. Figure 2 shows a graph of Factor X (FX) activation after treatment with single-chain or multi-chain chimeric polypeptides. Clotting times of buffers with various concentrations of Innovin in the prothrombin time (PT) test. Clotting times of multi-chain chimeric polypeptides in PT assay are shown. Figure 2 shows clotting times of multi-chain chimeric polypeptides in the PT assay when mixed with 32DB cells. Clotting times of multichain chimeric polypeptides in the PT assay when mixed with human PBMCs are shown. Binding of 7t15-21s137L (long version) and 7t15-21s137L (short version) to CD137 (4.1BB). Figures 189A-189D show antibody detection of IL7, IL21, IL15, and 4.1BBL in 7t15-21s137L (long version) using ELISA. IL-15 activity of 7t15-21s137L (long version) and 7t15-21s137L (short version) as assessed by IL2Rαβγ-containing CTLL2 cell proliferation assay. Figures 191A-191C show human blood lymphocyte pStat5a responses in CD4 ⁺ CD25 ^high T _reg cells, CD4 ⁺ CD25 ^- T _con cells, or CD8 ⁺ T _con cells in response to treatment with 2t2 or IL2. Figure 191A shows the pSTAT5 response in CD4 ⁺ CD25 ^high T _reg cells. FIG. C191B shows the pSTAT5 response in CD4 ⁺ CD25 ^high T _con cells. FIG. 191C shows pSTAT5 responses in CD8 ⁺ T _con cells. Figures 192A-192E are a series of images showing that treatment with an IL-2-based molecule (2t2) can induce hair follicle formation after hair loss in a mouse model. Figure 192A is an image from a control mouse-only that was shaved followed by depilation and Figure 192B is an image from a mouse that received a low dose of IL-2 (1 mg/kg) after depilation. 192C-192E show images from mice dosed with 2t2 at 0.3 mg/kg, (FIG. 192C), 1 mg/kg (FIG. 192D), and (FIG. 192E) 3 mg/kg after depilation. Black arrows indicate anagen hair follicles that later extend into the dermis and promote hair growth. Total number of anagen follicles counted per 10 fields for each treatment group is shown. Graph showing different percentages of DNA demethylation in NK cells from two different donors (compared to unexposed NK cells) after expansion with 7t15-21s + anti-tissue factor (TF) antibody (IgG1) (50 nM). is. FIG. 4 is a series of graphs showing immunophenotype by peripheral blood analysis after 4 days of single dose treatment with TGFRt15-TGFRs. FIG. 4 is a series of graphs showing immunophenotype by peripheral blood analysis after 4 days of single dose treatment with TGFRt15-TGFRs. Graph showing β-Gal staining analysis by FACS 7 days after the second dose of TGFRt15-TGFRs. 7 is a series of graphs showing levels of markers of senescence in liver tissue determined using qPCR 7 days after the second dose with TGFRt15-TGFRs. 7 is a series of graphs showing levels of markers of senescence in renal tissue determined using qPCR 7 days after the second dose with TGFRt15-TGFRs. 7 is a series of graphs showing levels of skin tissue senescence markers determined using qPCR 7 days after the second dose with TGFRt15-TGFRs. 7 is a series of graphs showing lung tissue senescence marker levels determined using qPCR 7 days after the second dose with TGFRt15-TGFRs. A series of histological images showing β-Gal staining of kidney tissue 7 days after a second treatment with TGFRt15-TGFRs. Figures 203A-203C show that chemotherapy induces p21 ^CIP1 p21 senescence-related gene expression in C57BL/6 mice. FIG. 203A is an exemplary schematic showing an experimental therapeutic regimen. Figures 203B and 203C are graphs showing p21 ^CIP1 p21 expression in lung (B) and liver (C) tissues, respectively. FIG. 3 is a series of graphs showing immunophenotype and cell proliferation following treatment with IL-15-based agents at 3 days post-treatment. Figures 205A-205C are graphs showing that TGFRt15-TGFRs treatment reduces senescence-related gene expression in C57BL/6 mice. Graphs show p21 ^CIP1 p21 and CD26 expression in lung (A and B) tissues and p21 ^CIP1 p21 expression in liver (C) tissues, respectively. FIG. 3 is a series of graphs showing the percentage and proliferation of CD4 ⁺ , CD8 ⁺ , and Treg cells. 2 is a series of graphs showing the percentage and proliferation of NK, CD19 ⁺ , and mononuclear cells. Figures 208A-208C are graphs showing the assessment of senescence markers p21 ^CIP1 p21 and CD26 in lung and liver tissue. Figures 208A and 208B show lung p21 ^CIP1 p21 (A) and lung CD26 (B) senescence markers. FIG. 208C shows liver p21 ^CIP1 p21 senescence markers. Schematic representation of interactions between exemplary TGFβRII/IL-15RαSu and TGFβRII/TF/IL-15Mut proteins leading to TGFRt15*-TGFRs complexes. Schematic representation of interactions between exemplary TGFβRII/IL-15RαSu and TGFβRII/TF/IL-15Mut proteins. FIG. 211A is a graph showing the binding activity of TGFRt15-TGFRs to TGF-β1 and LAP. FIG. 211B is a graph showing the binding activity of TGFRII/Fc to TGF-β1 and LAP. FIG. 211C is a graph showing the binding activity of TGFRt15-TGFRs to TGF-β1 and LAP. FIG. 211D is a graph showing the binding activity of TGFRt15*-TGFRs to TGF-β1 and LAP. FIG. 211E is a graph showing the binding activity of TGFRt15-TGFRs, TGFRt15*-TGFRs, and 7t15-21s to CTLL-2 cells. FIG. 212A is a graph of the TGF-β1 blocking activity of TGFRt15-TGFRs and TGFRt15*-TGFRs. FIG. 212B is a graph of IL-15 biological activity of TGFRt15-TGFRs and TGFRt15*-TGFRs. FIG. 212C is a graph showing that TGF-β1, TGF-β2, and TGF-β3 each similarly inhibit IL-4-induced CTLL-2 proliferation in the absence of TGFRt15*-TGFRs. FIG. 212D is a graph showing that TGFRt15*-TGFRs significantly reversed TGF-β1 and TGF-β3 inhibition of IL-4-induced CTLL-2 cell proliferation. Figure 213A shows that the IL-15 domain of TGFRt15-TGFRs is largely undamaged after 10 days of incubation at 4°C, 25°C, or 37°C. Figure 213B shows that there is no major damage to the TGFβ-RII domain of TGFRt15-TGFRs after 10 days of incubation at 4°C, 25°C, or 37°C. FIG. 213C is a graph showing the TGF-β1 neutralizing activity of TGFRt15-TGFRs after 10 days of incubation at 4° C., 25° C., or 37° C. in human serum. FIG. 213D is a graph showing IL-15 activity of TGFRt15-TGFRs after 10 days of incubation at 4° C., 25° C., or 37° C. in human serum. Figure 214A is a graph showing cell-mediated cytotoxicity in assays using NK cells and the indicated constructs. FIG. 214B is a graph showing cell-mediated cytotoxicity in assays using PMBCs and the indicated constructs. Figure 214C is a graph showing intracellular granzyme B production in assays using NK cells and the indicated constructs. FIG. 214D is a graph showing intracellular granzyme B production in assays using PBMCs and the indicated constructs. Figure 214E is a graph showing interferon gamma production in assays using NK cells and the indicated constructs. Figure 214F is a graph showing interferon gamma production in assays using PMBCs and the indicated constructs. Figure 10 is a graph showing the pharmacokinetics (half-life, t _1/2 ) of TGFRt15-TGFRs evaluated in female C57BL/6 mice. Graph showing toxicity of TGFRt15-TGFRs in C57BL/6 mice. FIG. 10 is a graph showing anti-tumor activity of TGFRt15-TGFRs in C57BL/6 mouse melanoma model. FIG. 50 μl of bleomycin (2.5 mg/kg, single dose) was administered to 9-week-old C57BL6/j male mice via the oropharyngeal route, and TGFRt15-TGFRs was administered subcutaneously (3 mg/kg) 17 days after bleomycin treatment. TGFRt15-TGFRs activity at 200°C. Fasting plasma glucose levels of db/db mice after 4 days of treatment with TGFRt15-TGFRs or TGFRt15*-TGFRs are shown. Figures 220A-220C show TGFβ1-3 levels in db/db mice after 4 days of treatment with TGFRt15-TGFRs or TGFRt15*-TGFRs: TGFβ1 (Figure 220A), TGFβ2 (Figure 220B) and TGFβ3 (Figure 220C). Figures 221A-E show TGFRt15-TGFRs or TGFRt15*-TGFRs: blood NK cells (Figure 221A), blood Ki67 ⁺ NK cells (Figure 221B), blood granzyme B ⁺ (GzmB ⁺ ) (Figure 221C), blood Lymphocyte subsets of db/db mice 4 days after treatment with medium CD8 ⁺ (FIG. 221D) and blood CD8 ⁺ Ki67 ⁺ T cells (FIG. 221E) are shown. FIG. 222A shows the interaction of TGFRt15*-TGFRs or TGFRt15-TGFRs with potential TGFβ1 (SLC) or CD39 (control). FIG. 222B shows the interaction of TGFRt15*-TGFRs and TGFRII-Fc with potential TGFβ1. FIG. 10 is a graph showing Innovin's clotting time in the PT assay. FIG. Graph showing the coagulation time of TGFRt15-TGFRs in PT assay. Graphs showing gene expression of senescence markers PAI-1, IL-1α, IL6, and IL-1β in the kidney in short-term and long-term follow-up of young mice and aged mice treated with PBS or TGFRt15-TGFRs. This is a comparison. FIG. 4 is a graph showing gene expression of senescence markers IL-1α and IL6 in liver. Figure 2 shows protein expression of the senescence marker PAI-1 in the kidney. Graph showing that IL15SA (positive control) or TGFRt15*-TGFRs+IL15SA mediated an increase in the percentage of CD3 ⁺ CD8 ⁺ , CD3 ⁻ NK1.1 ⁺ , and CD3 ⁺ CD45 ⁺ immune cells in the blood, whereas TGFRt15 Treatment with *-TGFR had little or no effect on the percentages of these cell populations. Graph showing that IL15SA (positive control) or TGFRt15*-TGFRs+IL15SA mediated an increase in the percentage of CD3 ⁺ CD8 ⁺ , CD3 ⁻ NK1.1 ⁺ , and CD3 ⁺ CD45 ⁺ immune cells in the spleen, whereas TGFRt15*. - Treatment with TGFR had little or no effect on the percentages of these cell populations. Figure 230A shows gene expression of the senescence marker p21 in kidney and liver tissues after treatment with test substances. FIG. 230B shows gene expression of the 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 the senescence marker IL-6 in kidney and liver tissues after study treatment. FIG. 231A shows the percentage and proliferation of CD4 ⁺ , CD8 ⁺ , and Treg cells after treatment with the indicated agents. FIG. 231B shows the percentage and proliferation of NK, CD19 ⁺ , and mononuclear cells after treatment with the indicated agents. Figure 232A shows the assessment of gene expression of the senescence marker p21 in lung tissue of mice after treatment with chemotherapy and the indicated agents. Figure 232B shows assessment of gene expression of the senescence marker CD26 in lung tissue of mice after treatment with chemotherapy and the indicated agents. FIG. 232C shows evaluation of gene expression of the senescence marker p21 in liver tissue of mice after treatment with chemotherapy and the indicated agents. Figures 233A-B are graphs showing that TGFRt15-TGFRs treatment enhances immune cell proliferation, expansion and activation in the peripheral blood of B16F10 tumor-bearing mice. Graph showing that TGFRt15-TGFRs treatment reduces TGFβ levels in plasma of B16F10 tumor-bearing mice. Graph showing that TGFRt15-TGFRs treatment reduces pro-inflammatory cytokine levels in plasma of B16F10 tumor-bearing mice. TGFRt15-TGFRs treatment enhances NK and CD8 expansion in the spleen of B16F10 tumor-bearing mice. Figures 237A-B show that TGFRt15-TGFRs treatment enhances glycolytic activity of splenocytes from B16F10 tumor-bearing mice. Figures 238A-B show that TGFRt15-TGFRs treatment enhances mitochondrial respiration of splenocytes from B16F10 tumor-bearing mice. Figures 239A-B show that TGFRt15-TGFRs treatment enhances NK and CD8 immune cell infiltration (TIL) into tumors of B16F10 tumor-bearing mice. Histopathological analysis of tumors after TGFRt15-TGFRs treatment is shown, tumors showed less mitotic and necrotic activity after TGFRt15-TGFRs + TA99 antibody treatment. The mitotic index correlates with dividing cells and the presence of necrosis is a more aggressive feature and a measure of poor prognosis. FIG. 10 is a graph showing the combination of anti-PD-L1 antibody with TGFRt15-TGFRs+TA99 antibody and chemotherapy in B16F10 melanoma mouse model. FIG. 4 is a graph showing that the anti-tumor effect of TGFRt15-TGFRs in B16F10 melanoma mouse model depends on NK and CD8 T cells. 243A-B are graphs showing gene expression of senescence markers p21, IL-1α and IL6 in liver and lung tissues of tumor-bearing mice after chemotherapy. FIG. 10 is a graph showing gene expression induction of senescence markers p21, IL6, H2AX, and NK cell ligands Raele and ULBP1 by docetaxel treatment of B16F10 GFP cells. Shown are tumor infiltrating lymphocytes/day after 4 days of treatment in tumor-bearing mice. Figures 246A-B show flow cytometry analysis of tumor cells, immunotherapy-treated mice showed lower numbers of GFP-positive senescence at days 4 and 10 after treatment compared to PBS control group. Tumor cells were shown (Fig. 246A) and tumor cells seeded in 24-well plates were evaluated by fluorescence microscopy (Fig. 246B). TGFβ levels in the kidneys of mice after induction of renal injury with cisplatin and treatment with TGFRt15-TGFRs. Figures 248A-C show the toxic effects of repeated subcutaneous administration of TGFRt15-TGFRs in C57BL/6 mice. Changes in body weight are indicated by SD21 (Figure 248A). Spleen weights (Figure 248B) and blood counts and differentials (Figure 248C) are shown for mice at SD7 after one dose of TGFRt15-TGFRs and at SD21 after two doses. Plasma levels of TGF-β isoforms in mice after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg), TGFRt15*-TGFRs (3 mg/kg) are shown. 250A-B Glycolytic capacity (ECAR) (FIG. 250A) and mitochondrial respiratory capacity (OCR) (FIG. 250B) in splenocytes of mice after in vivo treatment with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs or IL15SA. shows the change in the ratio of 251A-B show glycolytic capacity (ECAR) (FIG. 251A) and mitochondrial respiratory capacity (OCR) (FIG. 251B) in mouse splenocytes after in vitro treatment with PBS, TGFRt15-TGFRs, or TGFRt15*-TGFRs. Shows percentage change. Figures 252A-E show changes in tumor growth and survival of B16F10 melanoma tumors in C57BL/6 mice after in vitro treatment with PBS, TGFRt15-TGFRs, or TGFRt15*-TGFRs. Tumor volume (Figure 252A) and mouse survival (based on tumor volume <4000 ^mm3 ) (Figure 252B) were assessed. As in FIG. 252A, mice were treated intraperitoneally with anti-CD8, anti-NK, or anti-CD8 and anti-NK Abs for 1 week to deplete immune cells prior to injection of B16F10 melanoma tumor cells. Tumor-bearing mice were then treated with PBS or 20 mg/kg TGFRt15-TGFRs on days 1 and 4 after tumor cell inoculation. Animal tumor volume (Figure 252C) and mouse survival (Figure 252D) were assessed. B16F10 tumor-bearing mice were treated with PBS or 20 mg/kg TGFRt15-TGFRs on days 1 and 7 after tumor inoculation (FIG. 252E). Eleven days after tumor inoculation, tumors were harvested and tumor-infiltrating NK1.1 ⁺ cells and CD8 ⁺ T cells were quantified by flow cytometry. Figures 253A-B show treatment effects on fasting plasma glucose (Figure 253A) and insulin (Figure 253B) levels in db/db mice receiving PBS (control) or TGFRt15-TGFRs. FIG. 254A shows the fold change in gene expression levels in the pancreas of db/db mice receiving TGFRt15-TGFRs compared to PBS controls. Figures 254B-D show the mean fold changes in pancreatic expression levels of SASP, senescence and beta-cell index genes, respectively, for db/db mice receiving TGFRt15-TGFRs compared to PBS controls. Figures 255A-B show multispectral images of pancreatic tissue sections from db/db mice treated with PBS (control) (Figure 255A) or TGFRt15-TGFRs (Figure 255B). Representative islets are shown, insulin + islet beta cells as OPAL-520, insulin ⁺ p21 ⁺ beta cells (seen as white cells in grayscale images) as OPAL-570 compared to PBS treated group ( Figure 255A), decreased in the TGRt15-TGFRs treated group (Figure 255B). Figures 255C and 255D show the levels of islet insulin ⁺ (Figure 255C) and islet insulin ⁺ p21 ⁺ (Figure 255D) cells in pancreatic tissue sections from db/db mice treated with PBS (control) or TGFRt15-TGFRs. Figures 256A-C show the effect of treatment on the percentage of circulating immune cell subsets in db/db mice receiving PBS (control) or TGFRt15-TGFRs. Percentage of Ki67-positive immune cells induced in the blood after subcutaneous treatment of cynomolgus monkeys with TGFRt15-TGFRs compared to PBS (vehicle). Spleens isolated from young (6 weeks old) and old (72 weeks old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15*-TGFRs (3 mg/kg) Extracellular acidification rate (ECAR) representing glycolytic function of cells is shown. Spleens isolated from young (6 weeks old) and old (72 weeks old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15*-TGFRs (3 mg/kg) Oxygen consumption rate (OCR) representing glycolytic mitochondrial respiration of cells is shown. Immune cells in the blood of young (6 weeks old) and old (72 weeks old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15*-TGFRs (3 mg/kg) Indicates the percentage of the subset. Shown are the percentages of immune cell subsets in the spleens of young (6 weeks old) and old (72 weeks old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs or TGFRt15*-TGFRs. Gene expression levels of IL1-α, IL1-β, IL-6, p21 and PAI-1 in the liver of aged mice given 1 or 2 doses of TGFRt15-TGFRs are shown. Inflammation scores of liver tissue of aged mice given 1 or 2 doses of TGFRt15-TGFRs are shown. 1 shows expression levels of IL1-α, IL1-β, IL-6, IL-8, TGF-β, PAI-1, collagen and fibronectin proteins in the liver of aged mice treated with TGFRt15-TGFRs once or twice. . β-galactosidase levels in liver tissue of aged mice 4 days after in vivo treatment with PBS or TGFRt15-TGFRs are shown. Survival curves of 72-week-old C57BL/6 mice after a single subcutaneous treatment with PBS or TGFRt15-TGFRs (3 mg/kg) are shown. Protein levels of SASP factors in livers of B16F10 tumor-bearing mice after chemotherapy and TGFRt15-TGFRs+TA99 therapy. Figure 268A-B shows CD8 + T cells (dpCD8) and TIS B16F10-GFP cells (Figure 268A) and NK ^{and CD8 + T} cell (Figure 268B) levels in tumors of mice after chemotherapy and TGFRt15-TGFRs + TA99 therapy. Effect of NK cell (dpNK) antibody depletion. Figures 269A-E show the anti-tumor activity and mechanism of action of TGFRt15-TGFRs + TA99 in combination with 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 change in tumor volume over time and at day 18 after combination treatment comprising TGFRt15-TGFRs + TA99 + anti-PD-L1 antibody after doxetaxel compared to PBS or chemotherapy alone treatment. Figures 269C and 269D show treatment effects on the percentage of tumor-infiltrating CD28 ⁺ CD8 ⁺ T cells and splenic IFNγ ⁺ CD8 ⁺ T cells on day 18. Figure 269E shows the effect of treatment on levels of NKG2D (MFI) in tumor-infiltrating CD8 ⁺ and CD8 ⁺ CD44- ^high T cells on day 18. 270A-D show changes in tumor growth and survival of SW1990 human pancreatic tumors in C57BL/6 scid mice after in vitro treatment with PBS, gemcitabine and nab-paclitaxel chemotherapy, TGFRt15-TGFRs, or TGFRt15-TGFRs plus chemotherapy. show. FIG. 270A shows an exemplary schematic for treating SW1990 human pancreatic tumors in a xenograft mouse model. Figures 270B and 270C show changes in tumor volume over time and at day 38, respectively, after combination treatment comprising TGFRt15-TGFRs plus chemotherapy compared to PBS or chemotherapy alone. FIG. 270D shows treatment effects on survival of SW1990 human pancreatic tumor-bearing mice.

DETAILED DESCRIPTION Killing spontaneous 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 decreased activation of TGF-β receptors. Alternatively, provided herein are methods of reducing the number of senescent cells. Also methods of reducing spontaneous 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. provided herein. Reducing levels of spontaneous and/or treatment-induced markers 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-beta receptors A method is also provided herein. Also a method of reducing spontaneous and/or treatment-induced senescent cell activity 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. provided herein. Levels of SASP factors derived from spontaneous and/or treatment-induced senescent cells in a subject, including administering to the subject a therapeutically effective amount of one or more agents that result in decreased activation of TGF-β receptors. Or methods of reducing activity are also provided herein.

Killing spontaneous and/or treatment-induced senescent cells or reducing the number of 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 Further provided herein is a method of causing. Also herein are methods of reducing spontaneous and/or treatment-induced accumulation of 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. Also herein are methods of reducing the levels of markers of spontaneous 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. provided in the book. Also herein are methods of reducing spontaneous and/or treatment-induced senescent cell activity in a subject comprising administering to the subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. provided. Levels of one or more SASP factors derived from spontaneous 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 activity and/or activity.

A method of treating an age-related disease or condition in a subject in need of treatment of an age-related disease or condition comprising administering to a subject identified as having an age-related disease or condition a therapeutically effective number of one or more Provided herein are methods comprising administering a therapeutically effective amount of a natural killer (NK) cell activator of and/or activated NK cells. A method of killing senescent cells or reducing the number of senescent cells in a subject in need of killing senescent cells or reducing the number of senescent cells, said subject comprising a therapeutically effective number of one or more active NK cells Also provided herein are methods comprising administering a therapeutically effective amount of a stimulator and/or activated NK cells. 1. A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time, comprising administering to said subject a therapeutically effective number of 1 Also provided herein are methods comprising administering therapeutically effective amounts of one or more natural killer (NK) cell activators and/or activated NK cells. A method of helping treat obesity in a subject in need of help treating obesity 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 comprising administering and/or activated NK cells.

Activated NK Cells Some embodiments of any of the methods described herein provide a subject (e.g., any of the exemplary subjects described herein) with a therapeutically effective administration of a number of activated NK cells (eg, human activated NK cells). Activated NK cells are, for example, CD25, CD69, MTOR-C1, SREBP1, IFN-γ, and granzymes (eg, granzyme B) compared to resting NK cells (eg, human resting NK cells). NK cells (eg, human NK cells) that exhibit increased expression levels of 2 or more (eg, 3, 4, 5, or 6) of For example, activated NK cells are, for example, CD25, CD69, MTOR-C1, SREBP1, IFN-γ, and granzymes (eg, granzyme B) compared to resting NK cells (eg, human activated NK cells). at least a 10% increase (e.g., at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase in the expression level of two or more (e.g., three, four, five, or six) of increase, at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase, at least 85% increase, at least 90% increase, at least 95% increase, at least 100% increase, at least 120% increase, at least 140% increase, at least 160% increase, at least 180% increase, at least 200% increase, at least 220% increase, at least 240% increase, at least 260% increase, at least 280% increase, or at least 300% increase).

In some embodiments, activated NK cells are, for example, CD25, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, compared to resting NK cells (eg, human activated NK cells). NKp46, NKG2D, CD16, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C, CCR7, CXCR3, L-selectin, CXCR1, CXCR2, CX3CR1, ChemR23, CXCR4, CCR5, S1P5, c- Kit, mTORC1 2 or more of (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) at least 10% increase (e.g., at least 15% increase, at least 20% increase, at least 25% increase, at least 30% increase) , at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase , at least 85% increase, at least 90% increase, at least 95% increase, at least 100% increase, at least 120% increase, at least 140% increase, at least 160% increase, at least 180% increase, at least 200% increase, at least 220% increase , at least a 240% increase, at least a 260% increase, at least a 280% increase, or at least a 300% increase).

For example, activated NK cells (e.g., human activated NK cells) have, e.g., CD25, CD69, mTORC1, SREBP1, IFN-γ, and granzyme ( For example, about 10% to about 500% increase, about 10% to about 450% increase in expression levels of two or more (eg, 3, 4, 5, or 6) of granzyme B) , about 10% increase to about 400% increase, about 10% increase to about 350% increase, about 10% increase to about 300% increase, about 10% increase to about 280% increase, about 10% increase to about 260% increase , about 10% increase to about 240% increase, about 10% increase to about 220% increase, about 10% increase to about 200% increase, about 10% increase to about 180% increase, about 10% increase to about 160% increase , about 10% increase to about 140% increase, about 10% increase to about 120% increase, about 10% increase to about 100% increase, about 10% increase to about 80% increase, about 10% increase to about 60% increase , about 10% to about 40% increase, about 10% to about 20% increase, 20% to about 500% increase, about 20% to about 450% increase, about 20% to about 400% increase, about 20% increase to about 350% increase, about 20% increase to about 300% increase, about 20% increase to about 280% increase, about 20% increase to about 260% increase, about 20% increase to about 240% increase, about 20% increase to about 220% increase, about 20% increase to about 200% increase, about 20% increase to about 180% increase, about 20% increase to about 160% increase, about 20% increase to about 140% increase, about 20% increase to about 120% increase, about 20% increase to about 100% increase, about 20% increase to about 80% increase, about 20% increase to about 60% increase, about 20% increase to about 40% increase, 40% increase to about 500% increase, about 40% increase to about 450% increase, about 40% increase to about 400% increase, about 40% increase to about 350% increase, about 40% increase to about 300% increase, about 40% increase to about 280% increase, about 40% increase to about 260% increase, about 40% increase to about 240% increase, about 40% increase to about 220% increase, about 40% increase to about 200% increase, about 40% increase to about 180% increase, about 40% increase to about 160% increase, about 40% increase to about 140% increase, about 40% increase to about 120% increase, about 40% increase to about 100% increase, about 40% increase to about 80% increase, about 40% increase to about 60% increase, 60% increase to about 500% increase, about 60% increase to about 450% increase, about 60% increase to about 400% increase, about 60 % increase to about 350% increase, about 60% increase to about 300% increase, about 60% increase to about 280% increase, about 60% increase to about 260% increase, about 60% increase to about 240% increase, about 60 % increase to about 220% increase, about 60% increase to about 200% increase, about 60% increase to about 180% increase, about 60% increase to about 160% increase, about 60% increase to about 140% increase, about 60 % increase to about 120% increase, about 60% increase to about 100% increase, about 60% increase to about 80% increase, 80% increase to about 500% increase, about 80% increase to about 450% increase, about 80% Increase to about 400% increase, about 80% increase to about 350% increase, about 80% increase to about 300% increase, about 80% increase to about 280% increase, about 80% increase to about 260% increase, about 80% Increase to about 240% increase, about 80% increase to about 220% increase, about 80% increase to about 200% increase, about 80% increase to about 180% increase, about 80% increase to about 160% increase, about 80% Increase to about 140% increase, about 80% increase to about 120% increase, about 80% increase to about 100% increase, 100% increase to about 500% increase, about 100% increase to about 450% increase, about 100% increase ~ about 400% increase, about 100% increase to about 350% increase, about 100% increase to about 300% increase, about 100% increase to about 280% increase, about 100% increase to about 260% increase, about 100% increase ~ about 240% increase, about 100% increase to about 220% increase, about 100% increase to about 200% increase, about 100% increase to about 180% increase, about 100% increase to about 160% increase, about 100% increase ~ About 140% increase, about 100% increase ~ About 120% increase, 120% increase ~ About 500% increase, About 120% increase ~ About 450% increase, About 120% increase ~ About 400% increase, About 120% increase ~ About 350% increase, about 120% increase to about 300% increase, about 120% increase to about 280% increase, about 120% increase to about 260% increase, about 120% increase to about 240% increase, about 120% increase ~ about 220% increase, about 120% increase to about 200% increase, about 120% increase to about 180% increase, about 120% increase to about 160% increase, about 120% increase to about 140% increase, 140% increase to about 500% increase, about 140% increase to about 450% increase, about 140% increase to about 400% increase, about 140% increase to about 350% increase, about 140% increase to about 300% increase, about 140% increase to about 280% increase, about 140% increase to about 260% increase, about 140% increase to about 240% increase, about 140% increase to about 220% increase, about 140% increase to about 200% increase, about 140% increase to about 180% increase, about 140% increase to about 160% increase, 160% increase to about 500% increase, about 160% increase to about 450% increase, about 160% increase to about 400% increase, about 160% increase to about 350 % increase, about 160% increase to about 300% increase, about 160% increase to about 280% increase, about 160% increase to about 260% increase, about 160% increase to about 240% increase, about 160% increase to about 220% % increase, about 160% increase to about 200% increase, about 160% increase to about 180% increase, 180% increase to about 500% increase, about 180% increase to about 450% increase, about 180% increase to about 400% increase, about 180% increase to about 350% increase, about 180% increase to about 300% increase, about 180% increase to about 280% increase, about 180% increase to about 260% increase, about 180% increase to about 240% increase, about 180% increase to about 220% increase, about 180% increase to about 200% increase, 200% increase to about 500% increase, about 200% increase to about 450% increase, about 200% increase to about 400% increase , about 200% increase to about 350% increase, about 200% increase to about 300% increase, about 200% increase to about 280% increase, about 200% increase to about 260% increase, about 200% increase to about 240% increase , about 200% increase to about 220% increase, 220% increase to about 500% increase, about 220% increase to about 450% increase, about 220% increase to about 400% increase, about 220% increase to about 350% increase, about 220% increase to about 300% increase, about 220% increase to about 280% increase, about 220% increase to about 260% increase, about 220% increase to about 240% increase, 240% increase to about 500% increase, about 240% increase to about 450% increase, about 240% increase to about 400% increase, about 240% increase to about 350% increase, about 240% increase to about 300% increase, about 240% increase to about 280% increase, about 240% increase to about 260% increase, 260% increase to about 500% increase, about 260% increase to about 450% increase, about 260% increase to about 400% increase, about 260% increase to about 350% increase, about 260 % increase to about 300% increase, about 260% increase to about 280% increase, 280% increase to about 500% increase, about 280% increase to about 450% increase, about 280% increase to about 400% increase, about 280% Increase to about 350% increase, about 280% increase to about 300% increase, 300% increase to about 500% increase, about 300% increase to about 450% increase, about 300% increase to about 400% increase, about 300% increase ~ about 350% increase, 350% increase to about 500% increase, about 350% increase to about 450% increase, about 350% increase to about 400% increase, 400% increase to about 500% increase, about 400% increase to about It can have a 450% increase, or a 400% increase to about a 500% increase.

In some embodiments, the activated NK cells are CD25, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, CD16, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5 , KIR3DS1, NKG2C, CCR7, CXCR3, L-selectin, CXCR1, CXCR2, CX3CR1, ChemR23, CXCR4, CCR5, S1P5, c-Kit, mTORC1 (e.g., three, four, five, 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) can further have from about a 10% increase to about a 500% increase (eg, or any of the subranges of this range described herein) in the expression level of (eg, resting NK cells (e.g. compared to human activated NK cells).

CD25, CD69, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, CD16, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, KIR3DS1, NKG2C, CCR7, CXCR3, L- selectins, Assays that can be used to determine expression levels of CXCR1, CXCR2, CX3CR1, ChemR23, CXCR4, CCR5, S1P5, c-Kit, mTORC1, MYC, SREBP1, IFN-γ, and granzymes (eg, granzyme B) Non-limiting examples of include, eg, immunoblotting, fluorescence-assisted cell sorting, enzyme-linked immunosorbent assay, and RT-PCR.

ヒトＣＤ２５ ｃＤＮＡ（配列番号２）

Non-limiting examples of commercially available ELISA assays that can be used to determine CD25 expression levels are available from Diaclone, Covalab Biotechnology, and Caltag Medsystems. The protein and cDNA sequences for mature human CD25 are shown below.
Mature human CD25 protein (SEQ ID NO: 1)

Human CD25 cDNA (SEQ ID NO:2)

ヒトＣＤ６９ ｃＤＮＡ（配列番号４）

Non-limiting examples of commercially available ELISA assays that can be used to determine CD69 expression levels are available from RayBiotech, Novus Biologicals, and Aviscera Bioscience. The protein and cDNA sequences for mature human CD69 are shown below.
Mature human CD69 protein (SEQ ID NO: 3)

Human CD69 cDNA (SEQ ID NO:4)

ヒトＣＤ５９ ｃＤＮＡ（配列番号６）

The protein and cDNA sequences for mature human CD59 are shown below.
Mature human CD59 protein (SEQ ID NO:5)

Human CD59 cDNA (SEQ ID NO: 6)

ヒトＣＤ３５２ ｃＤＮＡ（配列番号８）

The protein and cDNA sequences for mature human CD352 are shown below.
Mature human CD352 protein (SEQ ID NO: 7)

Human CD352 cDNA (SEQ ID NO:8)

ヒトＮＫｐ８０ ｃＤＮＡ（配列番号：１０）

The protein and cDNA sequences for mature human NKp80 are shown below.
Mature human NKp80 protein (SEQ ID NO: 9)

Human NKp80 cDNA (SEQ ID NO: 10)

ヒトＤＮＡＭ－１ ｃＤＮＡ（配列番号１２）

The protein and cDNA sequences for mature human DNAM-1 are shown below.
Mature human DNAM-1 protein (SEQ ID NO: 11)

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

ヒト２Ｂ４ ｃＤＮＡ（配列番号１４）

The protein and cDNA sequences for mature human 2B4 are shown below.
Mature human 2B4 protein (SEQ ID NO: 13)

Human 2B4 cDNA (SEQ ID NO: 14)

ヒトＮＫｐ３０ ｃＤＮＡ（配列番号１６）

The protein and cDNA sequences for mature human NKp30 are shown below.
Mature human NKp30 protein (SEQ ID NO: 15)

Human NKp30 cDNA (SEQ ID NO: 16)

ヒトＮＫｐ４４ ｃＤＮＡ（配列番号１８）

The protein and cDNA sequences for mature human NKp44 are shown below.
Mature human NKp44 protein (SEQ ID NO: 17)

Human NKp44 cDNA (SEQ ID NO: 18)

ヒトＮＫｐ４６ ｃＤＮＡ（配列番号２０）

The protein and cDNA sequences for mature human NKp46 are shown below.
Mature human NKp46 protein (SEQ ID NO: 19)

Human NKp46 cDNA (SEQ ID NO:20)

ヒトＮＫＧ２Ｄ ｃＤＮＡ（配列番号２２）

The protein and cDNA sequences for mature human NKG2D are shown below.
Mature human NKG2D protein (SEQ ID NO: 21)

Human NKG2D cDNA (SEQ ID NO:22)

ヒトＣＤ１６ａ ｃＤＮＡ（配列番号２４）

The protein and cDNA sequences for mature human CD16a are shown below.
Mature human CD16a protein (SEQ ID NO:23)

Human CD16a cDNA (SEQ ID NO:24)

ヒトＣＤ１６ｂ ｃＤＮＡ（配列番号２６）

The protein and cDNA sequences for mature human CD16b are shown below.
Mature human CD16b protein (SEQ ID NO:25)

Human CD16b cDNA (SEQ ID NO:26)

ヒトＫＩＲ２ＤＳ１ ｃＤＮＡ（配列番号２８）

The protein and cDNA sequences for mature human KIR2DS1 are shown below.
Human KIR2DS1 protein (SEQ ID NO:27)

Human KIR2DS1 cDNA (SEQ ID NO:28)

ヒトＫＩＲ２ＤＳ２ ｃＤＮＡ（配列番号：３０）

The protein and cDNA sequences for mature human KIR2DS2 are shown below.
Human KIR2DS2 protein (SEQ ID NO:29)

Human KIR2DS2 cDNA (SEQ ID NO:30)

ヒトＫＩＲ２ＤＳ３ ｃＤＮＡ（配列番号３２）

The protein and cDNA sequences for mature human KIR2DS3 are shown below.
Mature human KIR2DS3 protein (SEQ ID NO:31)

Human KIR2DS3 cDNA (SEQ ID NO:32)

ヒトＫＩＲ２ＤＬ４ ｃＤＮＡ（配列番号３４）

The protein and cDNA sequences for mature human KIR2DL4 are shown below.
Mature human KIR2DL4 protein (SEQ ID NO:33)

Human KIR2DL4 cDNA (SEQ ID NO:34)

ヒトＫＩＲ２ＤＳ４ ｃＤＮＡ（配列番号３６）

The protein and cDNA sequences for mature human KIR2DS4 are shown below.
Mature human KIR2DS4 protein (SEQ ID NO:35)

Human KIR2DS4 cDNA (SEQ ID NO:36)

ヒトＫＩＲ２ＤＳ５ ｃＤＮＡ（配列番号３８）

The protein and cDNA sequences for mature human KIR2DS5 are shown below.
Mature human KIR2DS5 (SEQ ID NO:37)

Human KIR2DS5 cDNA (SEQ ID NO:38)

ヒトＫＩＲ３ＤＳ１ ｃＤＮＡ（配列番号４０）

The protein and cDNA sequences for mature human KIR3DS1 are shown below.
Mature human KIR3DS1 cDNA (SEQ ID NO:39)

Human KIR3DS1 cDNA (SEQ ID NO:40)

ヒトＮＫＧ２Ｃ ｃＤＮＡ（配列番号４２）

The protein and cDNA sequences for mature human NKG2C are shown below.
Mature human NKG2C protein (SEQ ID NO: 41)

Human NKG2C cDNA (SEQ ID NO:42)

ヒトＣＣＲ７ ｃＤＮＡ（配列番号４４）

The protein and cDNA sequences for mature human CCR7 are shown below.
Mature human CCR7 protein (SEQ ID NO: 43)

Human CCR7 cDNA (SEQ ID NO:44)

ヒトＣＸＣＲ３ ｃＤＮＡ（配列番号４６）

The protein and cDNA sequences for mature human CXCR3 are shown below.
Mature human CXCR3 protein (SEQ ID NO:45)

Human CXCR3 cDNA (SEQ ID NO:46)

ヒトＬ－セレクチンｃＤＮＡ（配列番号４８）

The protein and cDNA sequences for mature human L-selectin are shown below.
Mature human L-selectin protein (SEQ ID NO:47)

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

ヒトＣＸＣＲ１ ｃＤＮＡ（配列番号５０）

The protein and cDNA sequences for mature human CXCR1 are shown below.
Mature human CXCR1 protein (SEQ ID NO:49)

Human CXCR1 cDNA (SEQ ID NO:50)

ヒトＣＸＣＲ２ ｃＤＮＡ（配列番号５２）

The protein and cDNA sequences for mature human CXCR2 are shown below.
Mature human CXCR2 protein (SEQ ID NO: 51)

Human CXCR2 cDNA (SEQ ID NO:52)

ヒトＣＸ３ＣＲ１ ｃＤＮＡ（配列番号５４）

The protein and cDNA sequences for mature human CX3CR1 are shown below.
Mature human CX3CR1 protein (SEQ ID NO:53)

Human CX3CR1 cDNA (SEQ ID NO:54)

ヒトＣｈｅｍＲ２３ ｃＤＮＡ（配列番号５６）

The protein and cDNA sequences for mature human ChemR23 are shown below.
Mature human ChemR23 protein (SEQ ID NO:55)

Human ChemR23 cDNA (SEQ ID NO:56)

ヒトＣＸＣＲ４ ｃＤＮＡ（配列番号５８）

The protein and cDNA sequences for mature human CXCR4 are shown below.
Mature human CXCR4 protein (SEQ ID NO:57)

Human CXCR4 cDNA (SEQ ID NO:58)

ヒトＣＣＲ５ ｃＤＮＡ（配列番号６０）

The protein and cDNA sequences for mature human CCR5 are shown below.
Mature human CCR5 protein (SEQ ID NO:59)

Human CCR5 cDNA (SEQ ID NO: 60)

ヒトＳ１Ｐ５ ｃＤＮＡ（配列番号６２）

The protein and cDNA sequences for mature human S1P5 are shown below.
Mature human S1P5 protein (SEQ ID NO: 61)

Human S1P5 cDNA (SEQ ID NO:62)

ヒトＣ－ｋｉｔ ｃＤＮＡ（配列番号６４）

The protein and cDNA sequences for mature human C-kit are shown below.
Mature human C-kit protein (SEQ ID NO: 63)

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

ヒトｍＴＯＲ ｃＤＮＡ（配列番号６６）

The protein and cDNA sequences for mature human mTOR are shown below.
Mature human mTOR protein (SEQ ID NO: 65)

Human mTOR cDNA (SEQ ID NO: 66)

Non-limiting examples of commercially available ELISA assays that can be used to determine the expression level of SREBP1 are available from Novus Biologicals and Abcam. The protein and cDNA sequences for mature human SREBP1 are shown below.
Mature human SREBP1 protein (SEQ ID NO: 67)
MDEPPFSEAALEQALGEPCDLDAALLTDDIEDMLQLINNQDSDFPGLFDPPYAGSGAGGTDPASPDTSSPGSLSPPPATLSSLEAFLSGPQAAPSPLSPPQPAPTPLKMYPSMPAFSPGPGIKEESVPLSILQTPTPQPLPGALLPQSFPAPAPPQFSSTP VLGYPSPPGGFSTGSPPGNNTQQPLPGLPLASPPGVPPVSLHTQVQSVVPQQLLTVTAAPTAAPVTTTVTSQIQQVPVLLQPHFIKADSLLLTAMKTDGATVKAAGLSPLVSGTTVQTGPLPTLVSGGTILATVPLVVDAEKLPINRLAAG SKAPASAQSRGEKRTAHNAIEKRYRSSINDKIIELKDLVVGTEAKLNKSAVLRKAIDYIRFLQHSNQKLKQENLSLRTAVHKSKSLKDLVSACGSGGNTDVLMEGVKTEVEDTLTPPPSDAGSPFQSSPLSLGSRGSGSGGSGSDSEPDSPV FEDSKAKPEQRPSLHSRGMLDRSRLALCTLVFLCLSCNPLASLLGARGLPSPPSDTTSVYHSPGRNVLGTESRDGPGWAQWLLPPVVWLLNGLLVLVSLVLLFVYGEPVTRPHSGPAVYFWRHRKQADLDLARGDFAQAAQQLWLALRAL GRPLPTSHLDLACSLLWNLIRHLLQRLWVGRWLAGRAGGLQQDCALRVDASASARDAALVYHKLHQLHTMGKHTGGHLTATNLALSALNLAECAGDAVSVATLAEIYVAAALRVKTSLPRALHFLTRFFLSSARQACLAQSGSVPPA MQWLCHPVGHRFFVDGDWSVLSTPWESLYSLAGNPVDPLAQVTQLFREHLLERALNCVTQPNPSPGSADGDKEFSDALGYLQLLNSCSDAAGAPAYSFSISSSMATTTGVDPVAKWWASLTAVVIHWLRRDEEAAERLCPLVEHLPR VLQESERPLPRAALHSFKAARALLGCAKAAESGPASLTICEKASGYLQDSLATTPASSSIDKAVQLFLCDLLLLVVRTSLWRQQPPAPAPAAQGTSSRPQASALELRGFQRDLSSLRRLLAQSFRPAMRRVFLHEATARLMMAGASPTRTHQLLDRSLR RRAGPGGKGGAVAELEPRPTRREHAEALLLASCYLPPGFLSAPGQRVGMLAEAARTLEKLGDRRLLHDCQQMLMRLGGGTTVTSS
Human SREBP1 cDNA (SEQ ID NO: 68)

ヒトＩＦＮ－γ ｃＤＮＡ（配列番号７０）

Non-limiting examples of commercially available ELISA assays that can be used to determine IFN-γ expression levels are available from R&D Systems, Thermo Fisher Scientific, Abcam, Enzo Life Sciences, and RayBiotech. The protein and cDNA sequences for mature human IFN-γ are shown below.
Mature human IFN-γ (SEQ ID NO: 69)

Human IFN-γ cDNA (SEQ ID NO:70)

ヒトグランザイムＢ ｃＤＮＡ（配列番号７２）

Non-limiting examples of commercially available ELISA assays that can be used to determine granzyme B expression levels are available from RayBiotech, Thermo Fisher Scientific, and R&D Systems. The protein and cDNA sequences for mature human granzyme B are shown below.
Mature human granzyme B (SEQ ID NO: 71)

Human Granzyme B cDNA (SEQ ID NO:72)

ヒトＭｙｃ ｃＤＮＡ（配列番号３３０）

Non-limiting examples of commercially available ELISA assays that can be used to determine MYC expression levels are available from Invitrogen, LSBio, Biocodon Technologies, and Elisa Genie. The protein and cDNA sequences for mature human MYC are shown below.
Human Myc protein (SEQ ID NO:329)

Human Myc cDNA (SEQ ID NO:330)

In some embodiments, activated NK cells (e.g., human activated NK cells) are more effective than resting NK cells (e.g., human resting NK cells) in a subject (e.g., a subject described herein). any) or increased ability (e.g., at least 10% increase, at least 20% increase, at least 30% increase, at least 40%) to kill senescent cells (e.g., any of the senescent cells described herein) in vitro increase, at least 50% increase, at least 60% increase, at least 70% increase, at least 80% increase, at least 90% increase, at least 100% increase, at least 120% increase, at least 140% increase, at least 160% increase, at least 180% increase, at least 200% increase, at least 220% increase, at least 240% increase, at least 260% increase, at least 280% increase, or at least 300% increase).

In some embodiments, activated NK cells (e.g., human activated NK cells) undergo senescence in a subject (e.g., any of the subjects described herein) or in vivo. From about a 10% increase to about a 500% increase in the ability to kill a cell (e.g., any of the senescent cells described herein) (or any subrange of this range described herein) (compared to resting NK cells (eg, human resting NK cells)).

In some embodiments, activated NK cells (e.g., human activated NK cells) are, for example, antigen present on senescent cells or target cells compared to resting NK cells (e.g., human resting NK cells). an increase in cytotoxic activity in a contact cytotoxicity assay in the presence of an antibody that specifically binds to increase, at least 60% increase, at least 70% increase, at least 80% increase, at least 90% increase, at least 100% increase, at least 120% increase, at least 140% increase, at least 160% increase, at least 180% increase, at least 200% increase, at least 220% increase, at least 240% increase, at least 260% increase, at least 280% increase, or at least 300% increase).

In some embodiments, activated NK cells (e.g., human activated NK cells) are, for example, antigen present on senescent cells or target cells compared to resting NK cells (e.g., human resting NK cells). An increase in cytotoxic activity in a contact cytotoxicity assay in the presence of an antibody that specifically binds to the ).

In some embodiments, the activated NK cells are obtained by obtaining resting NK cells and contacting the resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators. contacting that results in the generation of activated NK cells that are then administered to the subject. In some examples of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some examples of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some examples of these methods, the resting NK cells are haploidentical resting NK cells. In some examples of these methods, the resting NK cells are allogeneic resting NK cells. In some examples of these methods, the resting NK cells are induced NK cells. In some examples of any of these methods, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

In some examples of these methods, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically defined liquid culture medium. Some examples of these methods include isolating activated NK cells (and optionally administering a therapeutically effective amount of the activated NK cells to a subject, e.g., a subject described herein). further administering to any of

In some embodiments of these methods, the contacting step is from about 2 hours to about 20 days (eg, from about 2 hours to about 18 days, from about 2 hours to about 16 days, from about 2 hours to about 14 days, from 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 ~ 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 hours to about 16 days, about 12 hours to about 14 days, about 12 hours to about 12 days, about 12 hours to about 10 days, about 12 hours to about 8 days, about 12 hours to about 7 days, about 12 hours to about 6 days, about 12 hours ~ about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 2 days, about 12 hours to about 1 day, about 1 day to about 18 days, about 1 day to about 16 days, about 1 to about 15 days, about 1 to about 14 days, about 1 to about 12 days, about 1 to about 10 days, about 1 to about 8 days, about 1 to about 7 days , about 1 to about 6 days, about 1 to about 5 days, about 1 to about 4 days, about 1 to about 3 days, about 1 to about 2 days, about 2 to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, about 2 days to about 8 days, about 2 days to about 7 days, about 2 days ~ about 6 days, about 2 days to about 5 days, about 2 days to about 4 days, about 2 days to about 3 days, about 3 days to about 18 days, about 3 days to about 16 days, about 3 days to about 14 days, about 3 days to about 12 days, about 3 days to about 10 days, about 3 days to about 8 days, about 3 days to about 7 days, about 3 days to about 6 days, about 3 days to about 5 days , about 3 days to about 4 days, about 4 days to about 18 days, about 4 days to about 16 days, about 4 days to about 14 days, about 4 days to about 12 days, about 4 days to about 10 days, about 4 days to about 8 days, about 4 days to about 7 days, about 4 days to about 6 days, about 4 days to about 5 days, about 5 days to about 18 days, about 5 days to about 16 days, about 5 days ~ about 14 days, about 5 days to about 12 days, about 5 days to about 10 days, about 5 days to about 8 days, about 5 days to about 7 days, about 5 days to about 6 days, about 6 days to about 18 days, about 6 days to about 16 days, about 6 days to about 14 days, about 6 days to about 12 days, about 6 days to about 10 days, about 6 days to about 8 days, about 6 days to about 7 days , about 7 days to about 18 days, about 7 days to about 16 days, about 7 days to about 14 days, about 7 days to about 12 days, about 7 days to about 10 days, about 7 days to about 8 days, about 8 days to about 18 days, about 8 days to about 16 days, about 8 days to about 14 days, about 8 days to about 12 days, about 8 days to about 10 days, about 9 days to about 18 days, about 9 days ~ about 16 days, about 9 days to about 14 days, about 9 days to about 12 days, about 12 days to about 18 days, about 12 days to about 16 days, about 12 days to about 14 days, about 14 days to about It is conducted over a period of 18 days, about 14 days to about 16 days, or about 16 days to about 18 days.

NK Cell Activators Methods are provided herein that involve the use or administration of one or more NK cell activators. In some embodiments, NK cell activators can be proteins. In some embodiments, the NK cell activator is 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 (e.g., exemplary type A and type B multi-chain chimeric polypeptides described herein), antibodies, recombinant cytokines or interleukins (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) can be In some embodiments, the NK cell activator is a small molecule (e.g., a glycogen synthase kinase-3 (GSK3) inhibitor, e.g., Cichocki et al., Cancer Res. 77:5664-5675, 2017). CHIR99021), or an aptamer.

In some embodiments of any of the one or more NK cell activating agents provided herein, at least one of the one or more NK cell activating agents comprises one or more IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL compared to levels of activation in the absence of NK cell activators -21 receptor, IL-33 receptor, CD16, CD69, CD25, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, and one or more (e.g., 2, 3, 4, 5, 6, 7, or 8) of KIR3DS1 (e.g., in immune cells, e.g., 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 IL-2 receptor activation specifically binds to soluble IL-2 or IL-2 receptor It is an agonistic antibody.

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

In some embodiments, at least one of the one or more NK cell activators that result in IL-12 receptor activation specifically binds to soluble IL-12 or IL-12 receptor It is an agonistic antibody.

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

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, at least one of the one or more NK cell activators that result in activation of NKG2D is an agonistic antibody that specifically binds NKG2D.

In some embodiments, at least one of the one or more NK cell activators that result in activation of KIR2DS1 is an agonistic antibody that specifically binds KIT2DS1.

In some embodiments, at least one of the one or more NK cell activators that result in activation of KIR2DS2/3 is an agonistic antibody that specifically binds KIT2DS2/3.

In some embodiments, at least one of the one or more NK cell activators that result in activation of KIR2DL4 is an agonistic antibody that specifically binds KIT2DL4.

In some embodiments, at least one of the one or more NK cell activators that result in activation of KIR2DS4 is an agonistic antibody that specifically binds KIT2DS4.

In some embodiments, at least one of the one or more NK cell activators that result in activation of KIR2DS5 is an agonistic antibody that specifically binds KIT2DS5.

In some embodiments, at least one of the one or more NK cell activators that result in activation of KIR3DS1 is an agonistic antibody that specifically binds KIT3DS1.

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 (e.g., two, 3, 4, or 5) compared to activation levels in the absence of one or more NK cell activators, PD-1, TGF-β receptor, TIGIT, CD1, TIM- 3, Siglec-7, IRP60, Tactile, IL1R8, NKG2A/KLRD1, KIR2DL1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2 (e.g. immune cells, e.g. human immune cells, for example, in human NK cells).

In some embodiments, at least one of the one or more NK cell activators that result in decreased TGF-β receptor activation is specifically directed to the soluble TGF-β receptor, TGF-β A binding antibody, 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 decreased activation of TIGIT is an antagonist antibody that specifically binds TIGIT, soluble TIGIT, or a ligand of TIGIT. It is an antibody that specifically binds.

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

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

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

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

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

In some embodiments, at least one of the one or more NK cell activators that result in decreased activation of IL1R8 is an antagonist antibody that specifically binds IL1R8, soluble IL1R8, or a ligand of IL1R8. It is an antibody that specifically binds.

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

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

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

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

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

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

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

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

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

ヒト可溶性ＰＤ－Ｌ１（配列番号７４）

ヒト可溶性ＴＩＧＩＴ（配列番号７５）

ヒト可溶性ＣＤ１Ａ（配列番号７６）

ヒト可溶性ＴＩＭ３（配列番号７７）

In some examples, the NK cell activator can 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 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 to SEQ ID NO:73 It can include sequences that are % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

In some embodiments, the soluble PD-L1 protein 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 to SEQ ID NO:74 It can include sequences that are % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

In some embodiments, the soluble TIGIT protein 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 to SEQ ID NO:75 , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

In some embodiments, the soluble CD1A protein 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 to SEQ ID NO:76 , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

In some embodiments, the soluble TIM3 protein 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 to SEQ ID NO:77 , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

Recombinant Antibodies In some examples, the NK activator is an agonistic antibody that specifically binds to the IL-2 receptor (see, for example, Gaulton et al., Clinical Immunology and Immunopathology 36(1):18-29, 1985). described), agonistic antibodies that specifically bind to the IL-7 receptor, agonistic antibodies that specifically bind to the IL-12 receptor (e.g., 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 (e.g., US Pat. Agonist antibodies that specifically bind to the IL-33 receptor (see, for example, those described in US Patent Application Publication No. 2007/160579). ), antagonist antibodies that specifically bind to PD-1 (see, for example, those described in US Patent Application Publication No. 7,521,051), antibodies that specifically bind to PD-L1 (for example, US Pat. No. 8,217,149), antibodies that specifically bind to TGF-β receptors, antagonist antibodies that specifically bind to TGF-β receptors (see, for example, European Patent Antagonist antibodies that specifically bind to TIGIT (see, for example, those described in WO2017/053748), antibodies that specifically bind to ligands of TIGIT (see, for example, those described in WO2011/127324), agonistic antibodies that specifically bind to CD1 (see, for example, Szalay et al., J. Immunol. 162(12):6955-6958, 1999). antibodies that specifically bind to ligands of CD1 (see, for example, those described in Kain et al., Immunity 41(4):543-554, 2014), specific for TIM-3 antagonist antibodies that specifically bind to ligands of TIM-3 (see, for example, those described in US Patent Application Publication No. 2015/218274), antibodies that specifically bind to ligands of TIM-3 (for example, US Patent Application Publication No. 2017/ 283499), agonistic antibodies that specifically bind to CD69 (eg, 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 (e.g., Yigit et al., Oncotarget 7 : 26346-26360, 2016), agonist antibodies that specifically bind to NKp80 (see, for example, Peipp et al., Oncotarget 6:32075-32088, 2015), Agonist antibodies that specifically bind to DNAM-1, agonist antibodies that specifically bind to 2B4 (see, for example, those described in Sandusky et al., European J. Immunol. 36:3268-3276, 2006) , an agonistic antibody that specifically binds to NKp30 (see, for example, those described in Kellner et al., OncoImmunology 5:1-12, 2016), an agonistic antibody that specifically binds to NKp44, an agonistic antibody that specifically binds to NKp46 Agonist antibodies that bind (see, for example, those described in Xiong et al., J. Clin. Invest. 123:4264-4272, 2013), agonist antibodies that specifically bind to NKG2D (for example, Kellner et al. ., OncoImmunology 5:1-12, 2016), agonistic antibodies that specifically bind to KIR2DS1 (see, for example, Xiong et al., J. Clin. Invest. 123:4264-4272, 2013). described), agonistic antibodies that specifically bind to KIR2Ds2/3 (see, for example, those described in Borgerding et al., Exp. Hematology 38:213-221, 2010), specific for KIR2DL4 agonistic antibodies that specifically bind to KIR2DS4 (see, for example, those described in Miah et al., J. Immunol. 180:2922-32, 2008), agonistic antibodies that specifically bind to KIR2DS4 (for example, Czaja et al. ., Genes and Immunity 15:33-37, 2014), agonistic antibodies that specifically bind to KIR2DS5 (eg, Czaja et al. , Genes and Immunity 15:33-37, 2014), agonistic antibodies that specifically bind to KIR3DS1 (for example, as described in Czaja et al., Genes and Immunity 15:33-37, 2014). antagonist antibodies that specifically bind to Siglec-7 (see, for example, those described in Hudak et al., Nature Chemical Biology 10:69-75, 2014), specifically bind to IRP60 (see, eg, Bachelet et al., J. Biol. Chem. 281:27190-27196, 2006), antagonist antibodies that specifically bind to Tactile (eg, Brooks et al. , Eur. J. Cancer61 (Suppl.1): S189, 2016), antagonist antibodies that specifically bind to IL1R8 (e.g., Molgora et al., Frontiers Immunol. 7:1, 2016). ), an antagonist antibody that specifically binds to NKG2A/KLRD1 (see, for example, Kim et al., Infection Immunity 76:5873-5882, 2008), specific for KIR2DL1 antagonist antibodies that specifically bind to KIR2DL2/3 (see, for example, those described in Weiner et al., Cell 148:1081-1084, 2012), antagonist antibodies that specifically bind to KIR2DL2/3 (see, for example, Weiner et al., Cell 148:1081-1084, 2012), antagonist antibodies that specifically bind to KIR2DL5 (see, for example, those described in US 9,067,997), and KIR3DL1 specifically. Antagonist antibodies that bind (see, for example, those described in US 9,067,997), antagonist antibodies that specifically bind to KIR3DL2 (see, for example, those described in US 9,067,997), ILT2 /LIR-1 (see, for example, that described in US Pat. No. 8,133,485) and an antagonist antibody that specifically binds LAG-2.

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

ヒト可溶性ＩＬ－７（配列番号７９）

ヒト可溶性ＩＬ－１２サブユニットアルファ（配列番号８０）

ヒト可溶性ＩＬ－１２サブユニットベータ（配列番号８１）

ヒト可溶性ＩＬ－１５（配列番号８２）

ヒト可溶性ＩＬ－２１（配列番号８３）

ヒト可溶性ＩＬ－３３（配列番号８４）

Recombinant Interleukins or Cytokines In some examples, the NK activator is, for example, soluble IL-2, soluble IL-7, soluble IL-12, soluble IL-15, soluble IL-21, and soluble IL-33 can be 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 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 to SEQ ID NO:78. It can include sequences that are % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

In some embodiments, the soluble IL-7 protein 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 to SEQ ID NO:79. It can include sequences that are % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

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

In some embodiments, the soluble IL-15 protein 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 to SEQ ID NO:82. It can include sequences that are % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

In some embodiments, the soluble IL-21 protein 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 to SEQ ID NO:83. It can include sequences that are % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

In some embodiments, the soluble IL-33 protein 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 to SEQ ID NO:84. It can include sequences that are % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical.

Soluble Cytokine or Interleukin Receptors In some examples of any of the soluble cytokines or interleukin receptors described herein, the soluble cytokine or interleukin receptor can be a soluble TGF-beta receptor. . In some examples, the soluble TGF-beta receptor is soluble TGF-beta receptor I (TGF-βRI) (see, eg, Docagne et al., Journal of Biological Chemistry 276(49):46243-46250, 2001). 194(9):1140-1151, 2016), soluble TGF-β receptor II (TGF-βRII) (eg, Yung et al., Am. J. Resp. Crit. Care Med. 194(9): 1140-1151, 2016). and soluble TGF-βRIII (see, eg, Heng et al., Placenta 57:320, 2017). In some instances, the soluble TGF-β receptor is a receptor “trap” for TGF-β (see, eg, Zwaagstra et al., Mol. Cancer Ther. 11(7):1477-1487, 2012). and those described in De Crescenzo et al., Transforming Growth Factor-β in Cancer Therapy, Volume II, pp 671-684).

Additional examples of soluble cytokines or soluble interleukin receptors are known in the art.

Non-limiting examples of single chain chimeric polypeptide NK cell activators include (i) a first target binding domain (e.g., one of the target binding domains described herein or known in the art) either), (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 A single chain chimeric polypeptide comprising a domain (eg, any of the target binding domains described herein or known in the art).

In some examples of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide is about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 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 amino acids, 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 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 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 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 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 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 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 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 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 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 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 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids 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 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 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 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids 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 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 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 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 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 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 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 400 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 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 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 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 amino acids, 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 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 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 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 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 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids 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 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 It can have a total length of 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., an exemplary any of the target binding domains) and the soluble tissue factor domain (eg, 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 comprises a first target binding domain (e.g., a any of the exemplary target binding domains known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). (eg, any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the single chain chimeric polypeptides described herein, a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) ) and the second target binding domain (eg, 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 comprises a soluble tissue factor domain (e.g., exemplary soluble tissue factor domain) and the second target binding domain (any of the exemplary target binding domains described herein or known in the art) (e.g., , any of the exemplary linker sequences 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 (e.g., an exemplary any of the target binding domains) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are immediately adjacent to each other. ing. In some embodiments of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide comprises a first target binding domain (e.g., a any of the exemplary target binding domains 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) or ) (eg, any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the single chain chimeric polypeptides described herein, a second target binding domain (e.g., an exemplary any of the target binding domains) and the soluble tissue factor domain (eg, 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 comprises a second target binding domain (e.g., a any of the exemplary target binding domains known in the art) and a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) ) (eg, any of the exemplary linker sequences described herein or known in the art).

In some embodiments, the single-chain chimeric polypeptide comprises
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGGSGGGGGSQVQLQQSGAELARPGASVK MSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKS GDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNNTNEFLIDVDKGEN YCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTT at least 70% identical (e.g., at least 75% identical to , at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
CAGATCGTGCTGACCCAAAGCCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCG CTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGAT CCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCCGGTCAAGGTTTAGAGTGGATCGGATATAT CAACCCTTCCCGGGGGCTACACCAACTATAACCAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTACTACTGCGCTAGGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGGTACC ACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAAATCCGGAGACTGGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGT GATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTTCCTACCCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCTACCATCCAGAGCTTCGAGCAAG TTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAAGACCGCTAAGACCAACACCAACGAGTTTTT ATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGG TGCTTCCGTGAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTTAACCTCCGACAA AAGCTCCATCACAGCCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGAC ATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCGGAAGCTCCCCTAAACTGTGCCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTA 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).

In some embodiments, the single-chain chimeric polypeptide comprises
MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSSYSLTISGMEAEDAATYYCQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGG SQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTTDKSSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILE WEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLIYTLYYWKSSSSG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSLTSEDSAL YYCARWGDGNYWGRGTTLTVSSGGGGSGGGGGSGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (SEQ ID NO: 87 ), 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).

In some embodiments, the single-chain chimeric polypeptide comprises
ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTATTCAGCTCCGCCTATTCCCAGATCGTGCTGACCCAAAGCCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAAC CAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCCTGAAGACGCTGCCACCTACTATTGCCAGCCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGC ACCAAGCTCGAAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATG CATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGGCTACACCAACTATAACCAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGGAGGACTCCGCTGTTACT ACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAG ATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTTCACACAACAGACACCGAGTGTGATTTAACCGACGAAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCTCTCTACGAGAATTCCCCCGA ATTCACCCTTATTTAGAGACCAATTTAGGCCAGCTCTACCATCCAGAGCTTCGAGCAAGTTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTG GAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGGC GAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTA CAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCCGGTGGGGGCGACGGCCAATTACTGGGGACGGGGGCCACAACACTGACCGTGAGCAGC GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCT CCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAA ACTGGAGACAAAGAGG (SEQ ID NO: 88), 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).

In some embodiments, the single-chain chimeric polypeptide comprises
VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGGSGGGGSGGGGSDIEMTQSPAIM SASLGERVTMTCTASSVSSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTD TECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTV NRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSSYSLTISGMEAEDAATYYCQWSSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGS 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).

In some embodiments, the single-chain chimeric polypeptide comprises
GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCCGGTCAAGGTTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACAC CAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGCACAAGAGCTCCATTACGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGGCGATGGCAATTATTGGGGCCGGGGGAACTACTTTTAACAGTGAGCTCCGGCG GCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTA AACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTCTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGGAGGCACAAAGCTGGAGA CCAAGCGGAGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCCACCAACTTCAAGACCATCCTCGAGTGGAGCCCAAGCCCCGTTAATCAAGTTTATACCGTGCAGATTTCCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCT CACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGGTGTTCTCCTACCCCGCTGGAACGTGGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCCGAATTCACCCCCTATCTGGAAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACA AAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAA GGGCGAGAACTACTGCTTCTCCGTGCAAGCCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGGAGAGTTTCGTGAGCAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGA CTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAG CCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGGTGGAGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGG CCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACA AGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC (SEQ ID NO: 90), and 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).

In some embodiments, the single-chain chimeric polypeptide comprises
MKWVTFISLLFLFSSAYSVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSG GGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSVSSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKRSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQ ISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVD KGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYCQQWSSNPFTFGSGTKLEIN RGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 91) 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).

In some embodiments, the single-chain chimeric polypeptide comprises
ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTATTTAGCAGCGCCTACAGCGTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAA GCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGCAAGAGCTCCATTACCGCTACATGGAGTTTTCCCTCTTAACTTCTGAGGACTCCGCTTTATACACTATTGCGCTCGTTGG GGCGATGGCAATTATTGGGGCCGGGGGAACTACTTTAACAGTGAGCTCCGGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGCGACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCT CCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCCAATTTAGCTAGCGGCGTTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTCTACTCTTTAACCATTAGCTCTATGGAGGCCCGAAGATGCCGCCACATACT TTTGCCATCAGTACCACCGGTCCCCTACCTTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGGGAGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCCAAGCCCCGTTAATCAAGTTTATACCGTGCAGATTTCCCAC CAAGAGCGGGCGACTGGAAATCCAAGTGCTTCTATACCAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGGTGTTCTCTCACCCCGCTGGAAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCC CTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCT CCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGGAGAGTTTCGTGAGCAGAT CGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCCGCTCACT TTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGTGGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGG TGGAGGTAGCCAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCCT CCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGGCCAAGGTACCACCTTAACA GTCTCCTCC (SEQ ID NO: 92), 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).

Some embodiments of any of the single-chain chimeric polypeptides described herein have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (eg, any of the exemplary target binding domains described herein or known in the art).

In some embodiments, the single-chain chimeric polypeptide has one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains at its N-terminus. (eg, any of the exemplary target binding domains described herein or known in the art). In some embodiments, one or more additional target binding domains at the N-terminus of the single-chain chimeric polypeptide (e.g., among exemplary target binding domains described herein or known in the art) 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 domains (e.g., any of the exemplary target binding domains described herein or known in the art), or soluble tissue factor domains (e.g., exemplary soluble tissue either of the factor domains). In some embodiments, the single-chain chimeric polypeptide comprises at least one additional target-binding domain (e.g., examples described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide. any of the exemplary target binding domains) and the 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), or a soluble tissue factor domain (e.g., described herein) a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the include.

In some embodiments of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide has one or more (e.g., 2, 3, 4, 5) at its C-terminus. , 6, 7, 8, 9, or 10) additional target binding domains (eg, any of the exemplary target binding domains described herein or known in the art). In some embodiments, one or more additional target binding domains at the C-terminus of the single-chain chimeric polypeptide (e.g., among exemplary target binding domains described herein or known in the art) a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), a second target A binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or a soluble tissue factor domain (e.g., described herein or known in the art) any of the known exemplary soluble tissue factor domains). In some embodiments, the single-chain chimeric polypeptide comprises at least one additional target-binding domain (e.g., examples described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide. any of the exemplary target binding domains) and the 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), or a soluble tissue factor domain (e.g., described herein) a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the include.

In some embodiments of any of the single chain chimeric polypeptides described herein, the single chain chimeric polypeptide has one or more (e.g., 2, 3) at its N-terminus and its C-terminus. , 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) including. In some embodiments, one or more additional antigen binding domains at the N-terminus of the single-chain chimeric polypeptide (e.g., among exemplary target binding domains described herein or known in the art) 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 domains (e.g., any of the exemplary target binding domains described herein or known in the art), or soluble tissue factor domains (e.g., exemplary soluble tissue either of the factor domains). In some embodiments, the single-chain chimeric polypeptide has one or more additional antigen binding domains at the N-terminus (e.g., one of the exemplary target binding domains described herein or known in the art). and a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), a second target A binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains) or ) between them (eg, any of the exemplary linker sequences described herein or known in the art). In some embodiments, the C-terminal one or more additional antigen binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) One, 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., or immediately adjacent to a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains) there is In some embodiments, the single chain chimeric polypeptide has one or more additional antigen binding domains at the C-terminus (e.g., one of the exemplary target binding domains described herein or known in the art). and 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 domains (e.g., any of the exemplary target binding domains described herein or known in the art), or soluble tissue factor domains (e.g., exemplary soluble tissue any of the factor domains), further comprising a linker sequence (eg, any of the exemplary linker sequences 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 (e.g., an exemplary any of the target binding domains), 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 Two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) specifically bind to the same antigen. In some embodiments, 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., described herein or known in the art) two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) of the exemplary target binding domains known in do. In some embodiments, 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., described herein or known in the art) Two or more (eg, 3, 4, 5, 6, 7, 8, 9, or 10) of the (any of the exemplary target binding domains known in ) comprise the same amino acid sequence.

In some embodiments of any of the single chain chimeric polypeptides described herein, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 ( 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., of exemplary target binding domains described herein or known in the art) any of them) each specifically binds the same antigen. In some embodiments, 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 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (eg, any of the exemplary target binding domains described herein or known in the art) each specifically bind the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional Each target binding domain contains the same amino acid sequence.

In some embodiments of any of the single chain chimeric polypeptides described herein, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 ( 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., of exemplary target binding domains described herein or known in the art) any of them) specifically bind to different antigens.

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

In some embodiments of any of the single chain chimeric polypeptides described herein, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) is 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2 , CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD , TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL -2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 It specifically binds to a target selected from the group consisting of the receptor, CD122 receptor, and CD28 receptor.

In some embodiments of any of the single chain chimeric polypeptides described herein, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 One or more of the additional target binding domains (eg, any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. 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, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 one or more of the additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukins or cytokine receptors . Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include 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.

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); a second target binding domain (e.g., any of the target binding domains described herein) and one or more target binding domains (e.g., any of the target binding domains described herein) ) are each independently CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36 , ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA -DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHC I ligand, scMHC II ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor , CD155 receptor, and CD122 receptor.

In some embodiments of any of the single chain chimeric polypeptides described herein, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 One or more of the additional target binding domains (eg, any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is 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, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 one or more of the additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukins or cytokine receptors . In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor soluble IL-4 receptor, or soluble IL-10 receptor.

Multichain Chimeric Polypeptide-Type A
Non-limiting examples of NK cell activators include (a) (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) the first of a pair of affinity domains , a first chimeric polypeptide, and (b) a second chimeric polypeptide comprising (i) a second of the pair of affinity domains and (ii) a second target binding domain. It is a chimeric polypeptide wherein a first chimeric polypeptide and a second chimeric polypeptide associate through binding of the first and second domains of a pair of affinity domains.

In some examples of any of the multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and/or the second chimeric polypeptide are each independently from about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 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 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 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 amino acids, about 100 amino acids to about 3000 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 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 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 420 amino acids amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 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 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 amino acids, 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 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 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, 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 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids 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 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 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 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids 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 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 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 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 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 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 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 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 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440 amino acids, about 400 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 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 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 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 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 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 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 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 1000 amino acids, about 700 amino acids to about 950 amino acids amino acids, from 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 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 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 multichain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein ) and a soluble tissue factor domain (eg, any of the exemplary soluble tissue factor domains described herein) are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide is a first target binding domain (herein any of the exemplary first target binding domains described in ) and a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). Further includes sequences (eg, 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, a 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 exemplary first domains of any of the exemplary pairs of affinity domains described herein) is the first directly adjacent to each other within one chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide comprises a soluble tissue factor domain (described herein) within the first chimeric polypeptide. ) and the first domain of the pair of affinity domains (e.g., any of the exemplary pair of affinity domains described herein). first domain) (eg, 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 domain of the pair of affinity domains (e.g., an exemplary affinity pair described herein any of the exemplary second domains of any of the domains) and a second target binding domain (e.g., any of the exemplary second target binding domains described herein) ) are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide is the second domain of the pair of affinity domains within the second chimeric polypeptide. (e.g., any of the exemplary second domains of any of the exemplary paired affinity domains described herein) and a second target binding domain (e.g., any of the exemplary second target binding domains described) and a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) further includes

In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide has one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 ) (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional antigen binding domains is a 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 a pair of affinity domains ( for example, any of the exemplary first domains of any of the exemplary pair of affinity domains described herein). In some embodiments, the first chimeric polypeptide comprises a soluble tissue factor domain (eg, any of the exemplary soluble tissue factor domains described herein) and one or more additional target-binding domains. a linker sequence (e.g., as described herein or any of the exemplary linker sequences known in the art), and/or one or more additional target binding domains (e.g., exemplary targets described herein or known in the art) binding domain) and the first domain of the pair of affinity domains (e.g., any of the exemplary pair of affinity domains described herein). a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the can further include

In some embodiments of any of the multichain chimeric polypeptides described herein, the first chimeric polypeptide has one or more at the N-terminus and/or C-terminus of the first chimeric polypeptide. (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains. In some embodiments, at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is the first domain of the pair of affinity domains within the first chimeric polypeptide (e.g., any exemplary pair of affinity domains described herein) directly adjacent to the first domain). In some embodiments, the first chimeric polypeptide comprises 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 domain of the pair of affinity domains (e.g., the exemplary affinity domain described herein of any of the exemplary pair of affinity domains described herein) any of the first domains) and a linker sequence (eg, any of the exemplary linker sequences described herein or known in the art). In some embodiments, at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) immediately flanks a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) within the first chimeric polypeptide . In some embodiments, the first chimeric polypeptide comprises 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) a linker sequence (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, one or more additional target binding domains (e.g., examples described herein or known in the art) at least one of the target binding domains (any of the specific target binding domains) is located at the N-terminus and/or C-terminus of the first chimeric polypeptide, 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 tissue factor domain within the first chimeric polypeptide (e.g., the present any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of a pair of affinity domains (e.g., the exemplary pair of any of the exemplary first domains of any of the affinity domains). In some embodiments, at least one additional target binding domain of the one or more additional target binding domains located at the N-terminus (e.g., examples described herein or known in the art) The first target binding domain in the first chimeric polypeptide (e.g., any of the exemplary target binding domains described herein or known in the art) any of the affinity domains described herein) or the first domain of a pair of affinity domains (e.g., an exemplary first domain described herein of any of an exemplary pair of affinity domains described herein). domain). In some embodiments, the first chimeric polypeptide comprises at least one additional target binding domain within the first chimeric polypeptide (e.g., exemplary domains described herein or known in the art). any of the target binding domains) and a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary paired affinity domains described herein) of further comprising a linker sequence (eg, any of the linker sequences described herein or known in the art) located in the . In some embodiments, at least one additional target binding domain of the one or more additional target binding domains located at the C-terminus (e.g., examples described herein or known in the art) The first target binding domain in the first chimeric polypeptide (e.g., any of the exemplary target binding domains described herein or known in the art) either) or the first domain of a pair of affinity domains (e.g., any exemplary first domain of any of the exemplary pair of affinity domains described herein) or ). In some embodiments, the first chimeric polypeptide comprises at least one additional target binding domain within the first chimeric polypeptide (e.g., exemplary domains described herein or known in the art). any of the target binding domains) and a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary paired affinity domains described herein) of (eg, any of the exemplary linker sequences described herein or known in the art) located in the . In some embodiments, a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and a first domain of a pair of affinity domains (e.g., or any of the exemplary paired affinity domains described herein) and one or more additional target binding domains ( For example, at least one of the exemplary target binding domains described herein or known in the art) is a soluble tissue factor domain, and/or the first of a pair of affinity domains. directly adjacent to one domain. In some embodiments, the first chimeric polypeptide comprises (i) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor described herein) and a soluble tissue factor domain ( e.g., any of the exemplary soluble tissue factor described herein) and the first domain of the pair of affinity domains (e.g., of the exemplary pair of affinity domains described herein) one or more additional target binding domains (e.g., any of the exemplary first domains described herein or known in the art) positioned between and/or (ii) the first domain of the pair of affinity domains and the soluble tissue factor domain and the first of the pair of affinity domains. One or more additional target binding domains positioned between one domain and at least one of the one or more additional target binding domains (e.g., the examples described herein or known in the art). any of the appropriate linker sequences).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide has one or more at the N-terminal and/or C-terminal end of the second chimeric polypeptide. (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., exemplary target binding domains described herein or known in the art) any of). In some embodiments, at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is the second domain of the pair of affinity domains within the second chimeric polypeptide (e.g., the exemplary second domain of any of the exemplary pair of affinity domains described herein directly adjacent to the In some embodiments, the second chimeric polypeptide comprises one or more additional target binding domains within the second chimeric polypeptide (e.g., exemplary domains described herein or known in the art). any of the exemplary affinity domain pairs described herein) and the second domain of the pair of affinity domains (e.g., any of the exemplary pair of affinity domains described herein). any of the exemplary second domains described herein) and a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) ) further includes. In some embodiments, at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) immediately flanks a second target binding domain (eg, any of the target binding domains described herein or known in the art) within the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide comprises one or more additional target binding domains within the second chimeric polypeptide (e.g., exemplary 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) further includes a linker sequence (eg, any of the exemplary linker sequences described herein or known in the art) between.

In some embodiments of any of the multichain chimeric polypeptides described herein, of the first target binding domain, the second target binding domain, and one or more additional target binding domains are specifically directed to the same antigen Join. In some embodiments, two or more (e.g., three or more, four or more, five more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or more) specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five , 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or more) 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 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 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 multichain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and one or more additional target binding domains are Binds specifically to different antigens. In some embodiments of any of the multichain chimeric polypeptides described herein, of the first target binding domain, the second target binding domain, and one or more additional target binding domains one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or more) of is a domain. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains (eg, scFvs or single domain antibodies).

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain), 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 (e.g., any of the exemplary target binding domains described herein or known in the art) of (e.g., 2, 3, 4, 5, 6) of , 7, 8, 9, or 10) is 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL- 2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor specifically binds to a target selected from the group consisting of receptors, CD122 receptors, and CD28 receptors.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain), 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 one or more of the target binding domains of (eg, any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. 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, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain), 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 (eg, any of the exemplary target binding domains described herein or known in the art) of is a soluble interleukin or cytokine receptor. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include 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.

In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain (e.g., any of the target binding domains described herein), the second 2 target binding domains (e.g., any of the target binding domains described herein) and one or more target binding domains (e.g., any of the target binding domains described herein) are each independently CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA- DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand , scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, It can specifically bind to a target selected from the group of CD155 receptor and CD122 receptor.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain), one or both of a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and 1 The one or more additional binding domains (eg, any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is 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 multichain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are soluble interleukins or cytokine receptors. is. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor soluble IL-4 receptor, or soluble IL-10 receptor.

Multichain Chimeric Polypeptides - Type B
A non-limiting example of an NK cell activator is (a) first and second chimeric polypeptides, each comprising (i) a first target binding domain, (ii) an Fc domain, and ( iii) first and second chimeric polypeptides comprising a first of a pair of affinity domains; and (b) third and fourth chimeric polypeptides, each comprising (i) a pair of a second domain of the affinity domain and (ii) third and fourth chimeric polypeptides comprising a second target binding domain, wherein the first and second chimeric polypeptides The peptide and the third and fourth chimeric polypeptides are associated through binding of the first and second domains of the pair of affinity domains, and the first and second chimeric polypeptides are associates through the Fc domain of

In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein ) and an Fc domain (eg, 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 are linked to the first target within the first and second chimeric polypeptides. Between a binding domain (any of the exemplary first target binding domains described herein) and an Fc domain (e.g., any of the exemplary Fc domains described herein) further includes a linker sequence (eg, 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, an Fc domain (e.g., any of the exemplary Fc domains described herein) and a pair of affinity A first domain of a sex domain (e.g., any of the exemplary first domains of any of an exemplary pair of affinity domains described herein) is a first and a second are immediately adjacent to each other within the chimeric polypeptide of . In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first and second chimeric polypeptides are Fc domains within the first and second chimeric polypeptides (this any of the exemplary Fc domains described herein) and the first domain of the pair of affinity domains (e.g., any of the exemplary pair of affinity domains described herein) any of the exemplary first domains) and a linker sequence (eg, 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 domain of the pair of affinity domains (e.g., an exemplary affinity pair described herein any of the exemplary second domains of any of the domains) and a second target binding domain (e.g., any of the exemplary second target binding domains described herein) ) are immediately adjacent to each other within 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 have a pair of affinities within the third and fourth chimeric polypeptides. A second domain of domains (e.g., any of the exemplary second domains of any of the exemplary pair of affinity domains described herein) and a second target binding domain ( e.g., any of the exemplary second target binding domains described herein) and a linker sequence (e.g., an exemplary linker sequence described herein or known in the art) any of).

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 the same antigen. In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind 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 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 are antigen binding domains (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 described exemplary second target binding domains). 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) ) include scFv or single domain antibodies.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA , B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor , stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16-binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor specifically binds to a target selected from

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are 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 multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain (e.g., as described herein or in the art) one or both of the exemplary target binding domains known in ) are soluble interleukins or cytokine receptors. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include 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.

In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each independently CD16a, CD33, CD20, CD19, CD22, CD123. , PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2 , CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD , TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell specific for a target selected from the group of factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor can be linked together.

In some embodiments of any of the multichain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are soluble interleukin or cytokine proteins. be. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is 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 multichain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are soluble interleukins or cytokine receptors. is. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor soluble IL-4 receptor, or soluble IL-10 receptor.

Tissue Factor Human tissue factor has three domains: (1) a 219 amino acid N-terminal extracellular domain (residues 1-219), (2) a 22 amino acid transmembrane domain (residues 220-242), and (3) ) is a 263 amino acid transmembrane protein containing a 21 amino acid cytoplasmic C-terminal tail (residues 242-263) ((UniProtKB identifier number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, It contains a single S-palmitoylation site.Deletion or mutation of the cytoplasmic domain was not found to affect tissue factor clotting activity.Tissue factor contains a single S-palmitoylation site within the intracellular domain of the protein at Cys245. -has a palmitoylation site Cys245 is located at the amino terminus of the intracellular domain and near the membrane surface The tissue factor transmembrane domain is composed of a single transmembrane α-helix.

The extracellular domain of tissue factor, composed of two fibronectin type III domains, is linked to the transmembrane domain via a 6 amino acid linker. This linker provides conformational flexibility to separate the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping β-sheets, with the upper sheet domain containing 3 antiparallel β-strands and the lower sheet containing 4 β-strands. ing. The β-strands are linked by β-loops between strands βA and βB, βC and βD, and βE and βF, all of which are conformationally conserved within the two modules. There are three short α-helical segments connecting the β-strands. A unique feature of tissue factor is a 17 amino acid β-hairpin between strands β10 and β11 that is not a common member of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is absent in the C-terminal domain and unique to tissue factor. Such fibronectin type III domain structures are characteristic of the immunoglobulin-like protein family fold and are conserved among a wide variety of extracellular proteins.

The zymogen FVII is rapidly converted to FVIIa by limited proteolysis when it binds to tissue to form an active tissue factor-FVIIa complex. FVIIa, which circulates as an enzyme at concentrations of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa on the tissue factor-FVIIa complex greatly increases the enzymatic activity of FVIIa, increasing the rate of hydrolysis of small chromogenic peptidyl substrates by approximately 20-100 fold and increasing the rate of hydrolysis of small chromogenic peptidyl substrates. Increases the rate of activation of the molecular substrates FIX and FX by nearly a million fold. Formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface) in concert with allosteric activation of the active site of FVIIa upon binding to tissue factor ) increases the activation rate of FIX or FX an additional 1,000-fold in a Ca ²⁺ -dependent manner. The approximately one million-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complexes compared to free FVIIa is a critical regulatory point in the coagulation cascade.

FVII is a single-chain polypeptide of approximately 50 kDa, consisting of 406 amino acid residues, containing an N-terminal γ-carboxyglutamate-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C It has a terminal serine protease domain. FVII is activated to FVIIa by specific proteolytic cleavage of the Ile- ¹⁵⁴ -Arg ¹⁵² bond in the short linker region between EGF2 and the protease domain. This cleavage causes the light and heavy chains to be held together by a single disulfide bond at Cys ¹³⁵ and Cys ²⁶² . FVIIa binds to phospholipid membranes in a Ca ²⁺ -dependent manner via its N-terminal GLA domain. Immediately C-terminal to the GLA domain is an aromatic stack and two EGF domains. An aromatic stack connects the GLA domain to an EGF1 domain that binds a single Ca ²⁺ ion. Occupancy of this Ca ²⁺ -binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consists of His ¹⁹³ , Asp ²⁴² and Ser ³⁴⁴ and binding of a single Ca ²⁺ ion within the FVIIa protease domain is essential for its catalytic activity. The proteolytic activity of FVII to FVIIa releases the newly formed amino terminus at Ile ¹⁵³ , folds back, inserts into the activation pocket and forms a salt bridge with the carboxylate of Asp ³⁴³ to form an oxy Generates anion holes. Formation of this salt bridge is essential for FVIIa activity. However, oxyanion hole formation does not occur with free FVIIa upon proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is important for FVIIa to its cognate substrates. Free FVIIa adopts a stable extended conformation when bound to the membrane with its active site positioned approximately 80 Å above the membrane surface. Upon binding of FVIIa to tissue factor, the FVa active site is repositioned approximately 6 Å closer to the membrane. This regulation may aid proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLA domain-less FVIIa, it was shown that the active site was still positioned at a similar distance from the membrane, indicating that tissue factor completely reversed FVIIa active site positioning even in the absence of FVIIa-membrane interaction. Show that you can help Additional data indicated that tissue factor supports full FVIIa proteolytic activity as long as the tissue factor extracellular domain is somehow tethered to the membrane surface. However, raising the active site of FVIIa beyond 80 Å from the membrane surface greatly reduced the ability of the tissue factor-FVIIa complex to activate FX, but not the tissue factor-FVIIa amidolytic activity. .

Alanine scanning mutagenesis has been used to assess the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010 , 1994, Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitution identified a limited number of residue positions where alanine substitution caused a 5-10 fold lower affinity for FVIIa binding. Most of these residue side chains were found to be fully exposed to solvent in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand binding site is located over a broad area of the boundary between the two modules. In the C-module, residues Arg ¹³⁵ and Phe ¹⁴⁰ , located on the protruding BC loop, provide independent contacts with FVIIa. Leu ¹³³ is located at the base of the finger-like structure and is packed in the gap between the two modules. This provides continuity to a major cluster of key binding residues consisting of Lys ²⁰ , Thr ⁶⁰ , Asp ⁵⁸ and Ile ²² . Thr ⁶⁰ is only partially solvent exposed and may play a local structural role rather than making significant contact with the ligand. The binding site extends on the concave side of the intermodular angle including ^Glu24 and ^Gln110 and potentially the more distal residue ^Val207 . The binding region extends from Asp58 to a convex region formed by Lys ⁴⁸ , Lys ⁴⁶ , Gln ³⁷ , Asp ⁴⁴ and Trp ⁴⁵ . Trp ⁴⁵ and Asp ⁴⁴ do not interact independently with FVIIa and the effect of mutations at Trp ⁴⁵ may be the structural importance of this side chain for the local packing of adjacent Asp ⁴⁴ and Gln ³⁷ side chains. It shows that it can reflect The interacting region further contains two surface-exposed aromatic residues Phe ⁷⁶ and Tyr ⁷⁸ that form part of the hydrophobic cluster within the N-module.

Known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, and certain proteinase-activated receptors. Mutational analysis revealed several residues that, when mutated, support full FVIIa amidolytic activity towards small peptidyl substrates but lack the ability to support activation of macromolecular substrates (i.e., FVII, FIX, and FX). (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9): 6375-6381, 1992; Huang et al., J Biol Chem 271 ( 36):21752-21757, 1996, Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region of residues 159-165, and residues within or adjacent to this flexible loop, have been shown to be essential for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate-binding exosite region of tissue factor that is well distant from the FVIIa active site. Replacing glycine residues with slightly more bulky alanine residues significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility provided by glycine is essential for the loop of residues 159-165 for tissue factor macromolecular substrate recognition.

It has also been demonstrated that residues Lys ¹⁶⁵ and Lys ¹⁶⁶ are important for substrate recognition and binding. Mutating any of these residues to alanine greatly reduces tissue factor cofactor function. Lys ¹⁶⁵ and Lys ¹⁶⁶ point outwards toward each other, with Lys ¹⁶⁵ pointing toward FVIIa in most tissue factor-FVIIa structures and Lys ¹⁶⁶ pointing toward the substrate-binding exosite region in crystal structures. . A putative salt bridge formation between FVIIa Lys ¹⁶⁵ and FVIIa Gla ³⁵ would support the notion that the interaction of tissue factor with the FVIIa GLA domain regulates substrate recognition. These results suggest that the C-terminal portion of the tissue factor ectodomain directly interacts with the GLA domains of FIX and FX, possibly adjacent EGF1 domains, and that the presence of the FVIIa GLA domain regulates these interactions either directly or indirectly. This suggests that it can be regulated by either target.

Soluble Tissue Factor Domain In some embodiments of any of the polypeptides, compositions, or methods described herein, the soluble tissue factor domain lacks a signal sequence, a transmembrane domain, and an intracellular domain. It can be a wild-type tissue factor polypeptide. In some instances, the soluble tissue factor domain can be a tissue factor variant, wherein the wild-type tissue factor polypeptide lacks the signal sequence, transmembrane domain, and intracellular domain and is further modified with selected amino acids. ing. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, soluble mouse tissue factor domains, soluble rat tissue factor domains, and variant soluble tissue factor domains are provided below.
Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO:93)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
Exemplary Nucleic Acid Encoding a Soluble Human Tissue Factor Domain (SEQ ID NO: 94) AGCGGCCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCGTTTACACAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTAT ACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCC AAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCA ACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
Exemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO:95)
agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtnlgqpviqqfeqdgrk lnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge
Exemplary Soluble Rat Tissue Factor Domain (SEQ ID NO:96)
Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtkigqpviqkyeqggtklkvt vkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge
Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO:97)
SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSL RDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO:98)
SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLR DVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

In some embodiments, the soluble tissue factor domain is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical to SEQ ID NO: 93, 95, 96, 97, or 98 , 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 , may include sequences that are at least 99% identical, or 100% identical. In some embodiments, the soluble tissue factor domain is 1-20 amino acids (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) were removed from its N-terminus, and/or 1-20 amino acids (eg, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its C-terminus. can include

As can be appreciated in the art, it is likely that amino acid variations that are conserved among different mammalian species will reduce protein activity and/or structural stability, while those skilled in the art will likely It will be appreciated that amino acid mutations that are not conserved among different mammalian species are unlikely to reduce protein activity and/or structural stability.

In some instances of any of the multichain chimeric polypeptides described herein, the soluble tissue factor domain is unable to bind Factor VIIa. In some examples of any of the multichain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

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

In some examples, the soluble tissue factor domain has a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein and a lysine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein. isoleucine, tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, mature wild-type human tissue factor protein tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. Does not contain one or more (eg, 2, 3, 4, 5, 6, or 7) of phenylalanine at a position. 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 examples, the soluble tissue factor domain 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 to SEQ ID NO:94 , at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical can be encoded by a nucleic acid comprising a sequence that is

In some embodiments, the soluble tissue factor domain is about 20 amino acids to about 220 amino acids, about 20 amino acids to about 215 amino acids, about 20 amino acids to about 210 amino acids, about 20 amino acids to about 205 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 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 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 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 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 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 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 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 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 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 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 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids 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 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 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 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 amino acids to about 205 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 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids 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 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 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 220 amino acids, about 180 amino acids to about 215 amino acids, about 180 amino acids to about 210 amino acids, about 180 amino acids to about 205 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 220 amino acids, about 185 amino acids to about 215 amino acids 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 amino acids, or from about 215 amino acids to about 220 amino acids.

Linker Sequences In some embodiments, a linker sequence can 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 examples, the linker sequence is a synthetic linker sequence.

In some embodiments of any of the single chain chimeric polypeptides described herein, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences ( For example, they may contain the same or different linker sequences, such as any of the exemplary linker sequences described herein or known in the art. In some embodiments of any of the single chain chimeric polypeptides described herein, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences ( For example, they may contain the same or different linker sequences, such as 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 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (eg, the same or different linker sequences, eg, 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 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (eg, the same or different linker sequences, eg, any of the exemplary linker sequences described herein or known in the art).

In some embodiments, the linker sequence is 1 amino acid to about 100 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 35 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 24 amino acids, 1 amino acid to about 22 amino acids amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 18 amino acids, 1 amino acid to about 16 amino acids, 1 amino acid to about 14 amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about 10 amino acids, 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 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 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 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 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 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 It can have the full length of amino acids.

In some embodiments, the linker is rich 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 rich in glycine and serine residues. In some embodiments, the linker comprises one or more glycine-serine residue pairs (GS), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more It has a GS pair. In some embodiments, the linker is one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO:99) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, or more GGGS (SEQ ID NO: 99) sequences. In some embodiments, the linker is one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGGGS (SEQ ID NO: 100) sequence. In some embodiments, the linker is one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 101) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, or more GGSG (SEQ ID NO: 101) sequences. In some embodiments, the linker comprises GGSSRSSSSSGGGGGSGGGG (SEQ ID NO:222).

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

Target Binding Domains In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and/or the additional one or more A target binding domain may 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., an exemplary soluble interleukin receptor or soluble cytokine receptor described herein) ).

In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and/or one or more additional target binding domains each independently from about 5 amino acids to about 1000 amino acids, from about 5 amino acids to about 950 amino acids, from about 5 amino acids to about 900 amino acids, from about 5 amino acids to about 850 amino acids, from about 5 amino acids to about 800 amino acids, from 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 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 260 amino acids, about 5 amino acids to about 240 amino acids, about 5 amino acids to about 220 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 195 amino acids, about 5 amino acids to about 190 amino acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 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 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 about 45 amino acids, about 5 amino acids to about 40 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 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 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 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 about 45 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 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 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 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 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 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 about 45 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 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 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 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 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 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 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 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 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 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 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 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 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 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 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 1000 amino acids 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 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 about 45 amino acids 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 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 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 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45 amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids, about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino acids, about 45 amino acids to about 300 amino acids, about 45 amino acids to about 280 amino acids, about 45 amino acids to about 260 amino acids, about 45 amino acids to about 240 amino acids, about 45 amino acids to about 220 amino acids, about 45 amino acids to about 200 amino acids, about 45 amino acids to about 195 amino acids, about 45 amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids, about 45 amino acids to about 120 amino acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 75 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 65 amino acids, about 45 amino acids to about 60 amino acids amino acids, about 45 amino acids to about 55 amino acids, about 45 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 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 65 amino acids, about 50 amino acids to about 60 amino acids 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 55 amino acids to 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 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 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 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 60 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 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 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 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 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 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 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 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 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 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 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 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 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 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids to about 135 amino acids, about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids , about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 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 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 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 amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130 amino acids , about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 1000 amino acids, about 110 amino acids to about 950 amino acids, about 110 amino acids to about 900 amino acids, about 110 amino acids to about 850 amino acids, 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 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 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 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 145 amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550 amino acids, about 145 amino acids to about 500 amino acids, about 145 amino acids to about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145 amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids, about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260 amino acids, about 145 amino acids to about 240 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 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 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids 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 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 to 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 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 1000 amino acids, about 170 amino acids to about 950 amino acids, about 170 amino acids to about 900 amino acids, about 170 amino acids to about 850 amino acids, about 170 amino acids to about 800 amino acids, about 170 amino acids to about 750 amino acids, about 170 amino acids to about 700 amino acids, about 170 amino acids to about 650 amino acids, about 170 amino acids to about 600 amino acids, about 170 amino acids to about 550 amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170 amino acids 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 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 to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to about 240 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 200 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 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 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 to about 300 amino acids, about 220 to about 280 amino acids, about 220 to about 260 amino acids, about 220 to about 240 amino acids, about 240 to about 1000 amino acids, about 240 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 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 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 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 45
0 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 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 to about 750 amino acids, about 550 to about 700 amino acids, about 550 to about 650 amino acids, about 550 to about 600 amino acids, about 600 to about 1000 amino acids, about 600 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 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 to about 1000 amino acids, about 750 to about 950 amino acids, about 750 to about 900 amino acids, about 750 to about 850 amino acids, about 750 to about 800 amino acids, about 800 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 It can have a total number of amino acids from to about 1000 amino acids, from about 900 amino acids to about 950 amino acids, or from about 950 amino acids to about 1000 amino acids.

Any of the target binding domains described herein are less than 1×10 ⁻⁷ M, less than 1×10 ⁻⁸ M, less than 1×10 ⁻⁹ M, less than 1×10 ⁻¹⁰ M, 1×10 It can bind its target with a dissociation equilibrium constant (K _D ) of less than ⁻¹¹ M, less than 1×10 ⁻¹² M, or less than 1×10 ⁻¹³ M. In some embodiments, the antigen binding protein constructs provided herein are about 1×10 ⁻³ M to about 1×10 ⁻⁵ M, about 1×10 ⁻⁴ M to about 1×10 ⁻⁶ M, about 1×10 ⁻⁵ M to about 1×10 ⁻⁷ M, about 1×10 ⁻⁶ M to about 1×10 ⁻⁸ M, about 1×10 ⁻⁷ M to about 1×10 ⁻⁹ M, binds to a particular antigen with a K _D of from about 1×10 ⁻⁸ M to about 1×10 ⁻¹⁰ M, or from about 1×10 ⁻⁹ M to about 1×10 ⁻¹¹ M, including these upper and lower limits can do.

Any of the target binding domains described herein are about 1 pM to about 30 nM (eg, about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM About 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM About 2 pM to about 950 pM, about 2 pM to about 900 pM, about 2 pM to about 850 pM, about 2 pM to about 800 pM, about 2 pM to about 750 pM, about 2 pM to about 700 pM, about 2 pM to about 650 pM, about 2 pM to about 600 pM, about 2 pM to about 550 pM, about 2 pM to about 500 pM, about 2 pM to about 450 pM, about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2 pM to about 150 pM about 2 pM to about 100 pM, about 2 pM to about 90 pM, about 2 pM to about 80 pM, about 2 pM to about 70 pM, about 2 pM to about 60 pM, about 2 pM to about 50 pM, about 2 pM to about 40 pM, about 2 pM to about 20 pM, about 2 pM to about 10 pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5 pM to about 15 nM, about 5 pM to about 10 nM, about 5 pM to about 5 nM, about 5 pM to about 2 nM, about 5 pM to about 1 nM, about 5 pM to about 950 pM, about 5 pM to about 900 pM, about 5 pM to about 850 pM, about 5 pM to about 800 pM About 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM to about 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30 pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM to about 30 nM, about 10 pM to about 25 nM About 10 pM to about 850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about 10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about 600 pM, about 10 pM to about 550 pM, about 10 pM to about 500 pM, about 10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about 350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about 10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 100 pM, about 10 pM to about 90 pM About 15 pM to about 25 nM, about 15 pM to about 20 nM, about 15 pM to about 15 nM, about 15 pM to about 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about 15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about 900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about 15 pM to about 750 pM, about 15 pM to about 700 pM, about 15 pM to about 650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about 15 pM to about 500 pM About 15 pM to about 90 pM, about 15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about 60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15 pM to about 30 pM, about 15 pM to about 20 pM, about 20 pM to about 30 nM, about 20 pM to about 25 nM, about 20 pM to about 20 nM, about 20 pM to about 15 nM, about 20 pM to about 10 nM, about 20 pM to about 5 nM, about 20 pM to about 2 nM, about 20 pM to about 1 nM, about 20 pM to about 950 pM About 20 pM to about 500 pM, about 20 pM to about 450 pM, about 20 pM to about 400 pM, about 20 pM to about 350 pM, about 20 pM to about 300 pM, about 20 pM to about 250 pM, about 20 pM to about 20 pM, about 200 pM to about 150 pM, about 20 pM to about 100 pM, about 20 pM to about 90 pM, about 20 pM to about 80 pM, about 20 pM to about 70 pM, about 20 pM to about 60 pM, about 20 pM to about 50 pM, about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM to about 30 nM About 30 pM to about 900 pM, about 30 pM to about 850 pM, about 30 pM to about 800 pM, about 30 pM to about 750 pM, about 30 pM to about 700 pM, about 30 pM to about 650 pM, about 30 pM to about 600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about 30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about 350 pM, about 30 pM to about 300 pM, about 30 pM to about 250 pM, about 30 pM to about 200 pM, about 30 pM to about 150 pM, about 30 pM to about 100 pM About 40 pM to about 30 nM, about 40 pM to about 15 nM, about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM to about 2 nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM, about 40 pM to about 900 pM, about 40 pM to about 850 pM, about 40 pM to about 800 pM, about 40 pM to about 750 pM, about 40 pM to about 700 pM, about 40 pM to about 650 pM, about 40 pM to about 600 pM, about 40 pM to about 550 pM, about 40 pM to about 500 pM, about 40 pM to about 450 pM About 40 pM to about 80 pM, about 40 pM to about 70 pM, about 40 pM to about 60 pM, about 40 pM to about 50 pM, about 50 pM to about 30 nM, about 50 pM to about 25 nM, about 50 pM to about 30 nM, about 50 pM to about 15 nM, about 50 pM to about 10 nM, about 50 pM to about 5 nM, about 50 pM to about 2 nM, about 50 pM to about 1 nM, about 50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about 850 pM, about 50 pM to about 800 pM, about 50 pM to about 750 pM About 50 pM to about 300 pM, about 50 pM to about 250 pM, about 50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about 100 pM, about 50 pM to about 90 pM, about 50 pM to about 80 pM, about 50 pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about 30 nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pM to about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM, about 60 pM to about 2 nM, about 60 pM to about 1 nM About 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM to about 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM, about 60 pM to about 300 pM, about 60 pM to about 250 pM, about 60 pM to about 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM, about 60 pM to about 90 pM, about 60 pM to about 80 pM, about 60 pM to about 70 pM, about 70 pM to about 30 nM, about 70 pM to about 25 nM, about 70 pM to about 30 nM, about 70 pM to about 15 nM About 70 pM to about 750 pM, about 70 pM to about 700 pM, about 70 pM to about 650 pM, about 70 pM to about 600 pM, about 70 pM to about 550 pM, about 70 pM to about 500 pM, about 70 pM to about 450 pM, about 70 pM to about 400 pM, about 70 pM to about 350 pM, about 70 pM to about 300 pM, about 70 pM to about 250 pM, about 70 pM to about 200 pM, about 70 pM to about 150 pM, about 70 pM to about 100 pM, about 70 pM to about 90 pM, about 70 pM to about 80 pM, about 80 pM to about 30 nM About 80 pM to about 900 pM, about 80 pM to about 850 pM, about 80 pM to about 800 pM, about 80 pM to about 750 pM, about 80 pM to about 700 pM, about 80 pM to about 650 pM, about 80 pM to about 600 pM, about 80 pM to about 550 pM, about 80 pM to about 500 pM, about 80 pM to about 450 pM, about 80 pM to about 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM, about 80 pM to about 250 pM,
about 80 pM to about 200 pM, about 80 pM to about 150 pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM, about 90 pM to about 30 nM, about 90 pM to about 25 nM, about 90 pM to about 30 nM, about 90 pM to about 15 nM, about 90 pM to about 10 nM, about 90 pM to about 5 nM, about 90 pM to about 2 nM, about 90 pM to about 1 nM, about 90 pM to about 950 pM, about 90 pM to about 900 pM, about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM to about 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM, about 90 pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM to about 500 pM, about 90 pM to about 450 pM, about 90 pM to about 400 pM, about 90 pM to about 350 pM, about 90 pM to about 300 pM, about 90 pM to about 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM, about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pM to about 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15 nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about 100 pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950 pM, about 100 pM to about 900 pM, about 100 pM to about 850 pM, about 100 pM to about 800 pM, about 100 pM to about 750 pM, about 100 pM to about 700 pM, about 100 pM to about 650 pM, about 100 pM to about 600 pM, about 100 pM to about 550 pM, about 100 pM to about 500 pM, about 100 pM to about 450 pM, about 100 pM to about 400 pM, about 100 pM to about 350 pM, about 100 pM to about 300 pM, about 100 pM to about 250 pM, about 100 pM to about 200 pM, about 100 pM to about 150 pM, about 150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM to about 30 nM, about 150 pM to about 15 nM, about 150 pM to about 10 nM, about 150 pM to about 5 nM, about 150 pM to about 2 nM, about 150 pM to about 1 nM, about 150 pM to about 950 pM, about 150 pM to about 900 pM, about 150 pM to about 850 pM, about 150 pM to about 800 pM, about 150 pM to about 750 pM, about 150 pM to about 700 pM, about 150 pM to about 650 pM, about 150 pM to about 600 pM, about 150 pM to about 550 pM, about 150 pM to about 500 pM, about 150 pM to about 450 pM, about 150 pM to about 400 pM, about 150 pM to about 350 pM, about 150 pM to about 300 pM, about 150 pM to about 250 pM, about 150 pM to about 200 pM, about 200 pM to about 30 nM, about 200 pM to about 25 nM, about 200 pM to about 30 nM, about 200 pM to about 15 nM, about 200 pM to about 10 nM, about 200 pM to about 5 nM, about 200 pM to about 2 nM, about 200 pM to about 1 nM, about 200 pM to about 950 pM, about 200 pM to about 900 pM, about 200 pM to about 850 pM, about 200 pM to about 800 pM, about 200 pM to about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650 pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM, about 200 pM to about 500 pM, about 200 pM to about 450 pM, about 200 pM to about 400 pM, about 200 pM to about 350 pM, about 200 pM to about 300 pM, about 200 pM to about 250 pM, about 300 pM to about 30 nM, about 300 pM to about 25 nM, about 300 pM to about 30 nM, about 300 pM to about 15 nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300 pM to about 2 nM, about 300 pM to about 1 nM, about 300 pM to about 950 pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM, about 300 pM to about 800 pM, about 300 pM to about 750 pM, about 300 pM to about 700 pM, about 300 pM to about 650 pM, about 300 pM to about 600 pM, about 300 pM to about 550 pM, about 300 pM to about 500 pM, about 300 pM to about 450 pM, about 300 pM to about 400 pM, about 300 pM to about 350 pM, about 400 pM to about 30 nM, about 400 pM to about 25 nM, about 400 pM to about 30 nM, about 400 pM to about 15 nM, about 400 pM to about 10 nM, about 400 pM to about 5 nM, about 400 pM to about 2 nM, about 400 pM to about 1 nM, about 400 pM to about 950 pM, about 400 pM to about 900 pM, about 400 pM to about 850 pM, about 400 pM to about 800 pM, about 400 pM to about 750 pM, about 400 pM to about 700 pM, about 400 pM to about 650 pM, about 400 pM to about 600 pM, about 400 pM to about 550 pM, about 400 pM to about 500 pM, about 500 pM to about 30 nM, about 500 pM to about 25 nM, about 500 pM to about 30 nM, about 500 pM to about 15 nM, about 500 pM to about 10 nM, about 500 pM to about 5 nM, about 500 pM to about 2 nM, about 500 pM to about 1 nM, about 500 pM to about 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850 pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about 500 pM to about 700 pM, about 500 pM to about 650 pM, about 500 pM to about 600 pM, about 500 pM to about 550 pM, about 600 pM to about 30 nM, about 600 pM to about 25 nM, about 600 pM to about 30 nM, about 600 pM to about 15 nM, about 600 pM to about 10 nM, about 600 pM to about 5 nM, about 600 pM to about 2 nM, about 600 pM to about 1 nM, about 600 pM to about 950 pM, about 600 pM to about 900 pM, about 600 pM to about 850 pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about 600 pM to about 700 pM, about 600 pM to about 650 pM, about 700 pM to about 30 nM, about 700 pM to about 25 nM, about 700 pM to about 30 nM, about 700 pM to about 15 nM, about 700 pM to about 10 nM, about 700 pM to about 5 nM, about 700 pM to about 2 nM, about 700 pM to about 1 nM, about 700 pM to about 950 pM, about 700 pM to about 900 pM, about 700 pM to about 850 pM, about 700 pM to about 800 pM, about 700 pM to about 750 pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM, about 800 pM to about 30 nM, about 800 pM to about 15 nM, about 800 pM to about 10 nM, about 800 pM to about 5 nM, about 800 pM to about 2 nM, about 800 pM to about 1 nM, about 800 pM to about 950 pM, about 800 pM to about 900 pM, about 800 pM to about 850 pM, about 900 pM to about 30 nM, about 900 pM to about 25 nM, about 900 pM to about 30 nM, about 900 pM to about 15 nM, about 900 pM to about 10 nM, about 900 pM to about 5 nM, about 900 pM to about 2 nM, about 900 pM to about 1 nM, about 900 pM to about 950 pM, about 1 nM to about 30 nM, about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30 nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nM to about 15 nM, about 15 nM about 30 nM, about 15 nM to _about 25 nM, about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nM to about 25 nM).

Any of the target binding domains described herein are about 1 nM to about 10 nM (eg, about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM About 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM, about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about 6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM to about 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM, about 6 nM to about 7 nM , about 7 nM to about 10 _nM , about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM). .

K _D values for any of the antigen binding protein constructs described herein can be determined using a variety of different methods known in the art (e.g., electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecule binding kinetic assays, etc.).

Antigen Binding Domains 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 specific for the same antigen. connect to each other. In some embodiments of these single-chain or multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these single-chain 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- or multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain specifically bind to different antigens. do.

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 antigen binding domain is. 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 are each antigen binding domains. In some embodiments of any of the single- or multi-chain chimeric polypeptides described herein, the antigen-binding domain is a scFv or single domain antibody (eg, Va _H H or V _NAR domains) contains or is

In some examples, the antigen binding domain (e.g., any of the antigen binding domains described herein) is CD16a (e.g., see those described in US Pat. No. 9,035,026). ), CD28 (see, for example, US Pat. No. 7,723,482), CD3 (see, for example, US Pat. No. 9,226,962), CD33 (see, for example, US Pat. No. 9,226,962). See, for example, US Pat. No. 8,759,494), CD20 (see, for example, WO2014/026054), CD19 (see, for example, US Pat. No. 9,701,758). CD22 (see, for example, WO2003/104425), CD123 (see, for example, WO2014/130635), IL-1R (for example, see , see, US Pat. No. 8,741,604), IL-1 (see, for example, WO2014/095808), VEGF (see, for example, US Pat. No. 9,090, 684), IL-6R (see, for example, US Pat. No. 7,482,436), IL-4 (see, for example, US Patent Application Publication No. 2012/ 0171197), IL-10 (see, e.g., US Patent Application Publication No. 2016/0340413), PDL-1 (e.g., Drees et al., Protein Express 94:60-66, 2014), TIGIT (see, eg, US Patent Application Publication No. 2017/0198042), PD-1 (see, eg, US See, for example, US Pat. No. 7,488,802), TIM3 (see, for example, US Pat. No. 8,552,156), CTLA4 (see, for example, WO2012/120125). ), MICA (see, for example, WO2016/154585), MICB (see, for example, US Pat. No. 8,753,640), IL- 6 (see, for example, Gejima et al., Human Antibodies 11(4):121-129, 2002), IL-8 (see, for example, US Pat. No. 6,117,980). see), TNFα (eg Geng et al. , Immunol. Res. 62(3):377-385, 2015), CD26 (see, e.g., WO2017/189526), CD36 (e.g., US Patent Application Publication No. 2015/0259429). ULBP2 (see, e.g., U.S. Patent No. 9,273,136), CD30 (e.g., Homach et al., Scand. J. Immunol. 48 ( 5):497-501, 1998), CD200 (see, for example, US Pat. No. 9,085,623), IGF-1R (see, for example, US patent applications). See, for example, Publication No. 2017/0051063), MUC4AC (see, for example, WO2012/170470), MUC5AC (for example, as described in U.S. Pat. No. 9,238,084). ), Trop-2 (see, eg, WO2013/068946), CMET (see, eg, Edwarddraja et al., Biotechnol. Bioeng. 106(3):367-375, 2010). EGFR (see, for example, Akbari et al., Protein Expr. Purif. 127:8-15, 2016), HER1 (see, for example, US Patent Application Publication No. 2013/ 0274446), HER2 (see, eg, Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3 (see, eg, US 9,505,843), PSMA (see, for example, Parker et al., Protein Expr. Purif. 89(2):136-145, 2013). ), CEA (see, for example, WO 1995/015341), B7H3 (see, for example, US Pat. No. 9,371,395), EPCAM (see, for example, WO 2014/159531). BCMA (see, for example, Smith et al., Mol. Ther. 26(6):1447-1456, 2018), P-cadherin (see, for example, US Patent 7,452,537), CEACAM5 (see, for example, US Pat. No. 9,617,345), UL16 binding proteins (see, for example, WO2017/083612). see description), HLA-DR (eg Pistillo et al. , Exp. Clin. Immunogenet. 14(2):123-130, 1997), DLL4 (see, for example, WO2014/007513), TYRO3 (see, for example, WO2016/166348), AXL (see, e.g., described in WO2012/175692), MER (see, e.g., described in WO2016/106221), CD122 (see, e.g., US Patent Application Publication No. 2016/0367664) ), CD155 (see, for example, WO2017/149538), or PDGF-DD (see, for example, US Pat. No. 9,441,034). can specifically bind to any one of them.

Each antigen-binding domain present in any of the single- or multi-chain chimeric polypeptides described herein is independently selected from the group consisting of VHH domains, VNAR domains, and scFv. In some embodiments, any of the antigen binding domains described herein are BiTe, (scFv) ₂ , Nanobody, Nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH- CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. Additional examples of antigen binding domains that can be used in either single-chain chimeric polypeptides or multi-chain chimeric polypeptides are known in the art.

VHH domains are single monomeric variable antibody domains that can be found in camelids. VNAR domains are single monomeric variable antibody domains that can be found in cartilaginous fish. Non-limiting aspects of V _H H domains and V _NAR domains are described, for example, in Cromie 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. , Expert. Opin. Biol. Ther. 14:1527-1539, 2014, Krah et al. , Immunopharmacol. Immunotoxicol. 38:21-28, 2016, Music-Delic et al. , Trends Pharmacol. Sci. 35:247-255, 2014; Biotechnol. 74:277-302, 2001, Muyldermans et al. , Trends Biochem. Sci. 26:230-235, 2001, Muyldermans, 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. Investig. 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 of the antigen binding domains within the single-chain or multi-chain chimeric polypeptides described herein are both VHH domains, or at least one antigen binding domain is a VHH domain . In some embodiments, each of the antigen binding domains within the single or multi-chain chimeric polypeptides described herein are both VNAR domains, or at least one antigen binding domain is a VNAR domain. . In some embodiments, each of the antigen binding domains of the single or multi-chain chimeric polypeptides described herein are both scFv domains, 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 are assembled (e.g., non-covalently assembled) to form an antigen described herein. Any of the binding domains, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), VHH-scAb, VHH-Fab, double scFab, F ( ab') 2, diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange , SEEDbody, LUZ-Y, Fcab, κλ-body, 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-CH3, Triplebody, Minobody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab- scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv1- PEG-scFv2 can be formed. For a description of these elements, see, for example, Spiess et al. , Mol. Immunol. 67:95-106, 2015. Non-limiting examples of antigen-binding fragments of antibodies include Fv, Fab, F(ab') ₂ , and Fab' fragments. Additional examples of antigen-binding fragments of antibodies include antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., human or humanized IgG, e.g., human or humanized IgG1, IgG2 , IgG3, or IgG4), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., human or humanized IgA, e.g., antigen-binding fragments of human or humanized IgA1 or IgA2) , an antigen-binding fragment of IgD (e.g., an antigen-binding fragment of human or humanized IgD), an antigen-binding fragment of IgE (e.g., an antigen-binding fragment of human or humanized IgE), or an antigen-binding fragment of IgM (e.g., human or antigen-binding fragment of humanized IgM).

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

A "Fab" fragment contains, in addition to the heavy and light chain variable domains of the Fv fragment, the constant domain of the light chain and the first constant domain of the heavy chain (C _H1 ).

An “F(ab′) ₂ ” fragment contains two Fab fragments joined by disulfide bonds near the hinge region.

A "dual variable domain immunoglobulin" or "DVD-Ig" is defined, for example, in DiGiammarino et al. , Methods Mol. Biol. 899:145-156, 2012, Jakob et al. , MABs 5:358-363, 2013, and U.S. Pat. , 722,855, 8,735,546, and 8,822,645.

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

Some embodiments of any of the antigen binding domains described herein are capable of binding antigens selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

Additional examples and embodiments of antigen binding domains are known in the art.

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ヒト可溶性ＩＬ－１７（配列番号１０８）

ヒト可溶性ＩＬ－１８（配列番号１０９）

ヒト可溶性ＰＤＧＦ－ＤＤ（配列番号１１０）

ヒト可溶性ＳＣＦ（配列番号１１１）

ヒト可溶性ＦＬＴ３Ｌ（配列番号１１２）

Soluble Interleukin or Cytokine Proteins In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one of the first target binding domain and the second target binding domain or Both can be soluble interleukin proteins or soluble cytokine proteins. In some embodiments, the soluble interleukin or soluble cytokine protein is 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-chain or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are Soluble interleukin receptors or soluble cytokine receptors. In some embodiments, the soluble receptor is soluble TGF-beta receptor II (TGF-beta RII) (eg, Yung et al., Am. J. Resp. Crit. Care Med. 194 (9) : 1140-1151, 2016), soluble TGF-βRIII (see, for example, Heng et al., Placenta 57:320, 2017), soluble NKG2D (see, for example, Cosman et al., Immunity 14(2): 123-133, 2001, Costa et al., Front. Immunol., Vol.9, Article 1150, May 29, 2018, doi: 10.3389/fimmu.2018.01150 see), soluble NKp30 (see, e.g., Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi: 10.3389/fimmu.2018.01150), soluble NKp44 ( See, for example, Costa et al., Front. Immunol., Vol.9, Article 1150, May 29, 2018, doi: 10.3389/fimmu. See Mandelboim et al., Nature 409:1055-1060, 2001, Costa et al., Front. ), soluble DNAM1 (see, for example, Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi: 10.3389/fimmu.2018.01150) , scMHC I (see, e.g., Washburn et al., PLoS One 6(3):e18439, 2011), scMHC II (e.g., Bishwajit et al., Cellular Immunol. 170(1):25-33). , 1996), scTCR (see, for example, Weber et al., Nature 356(6372):793-796, 1992), soluble CD155 (see, for example, Tahara-Hanaoka et al. , Int. Immunol. 16(4):533-538, 2004), or soluble CD28 (see, eg, Hebbar et al., Clin. Exp. Immunol. 136:388-392, 2004).

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

Pair of Affinity Domains In some embodiments, the multi-chain chimeric polypeptide comprises: 1) a first chimeric polypeptide comprising a first domain of a pair of affinity domains; and a second chimeric polypeptide comprising two domains, whereby the first chimeric polypeptide and the second chimeric polypeptide effect binding of the first and second domains of a pair of affinity domains. will meet through 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 type 1 glycoprotein motifs, are common motifs in protein-protein interactions. Sushi domains have been identified on several protein-binding molecules, including complement components C1r, C1s, factor H, C2m, and the non-immunological molecules factor XIII and β2-glycoprotein. A typical sushi domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp Biocomput. 2000:155-67). The first cysteine can form a disulfide bond with a third cysteine and the second cysteine can form a disulfide bridge with a fourth cysteine. In some embodiments where one member of a pair of affinity domains is soluble IL-15, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments in which one member of a pair of affinity domains is human IL-15 receptor alpha chain (IL-15Rα), human IL-15Rα is mature full-length IL-15Rα. In some embodiments, the pair of affinity domains is barnase and barnstar. In some embodiments, the paired affinity domains are PKA and AKAP. In some embodiments, the pair of affinity domains is a mutated RNase I fragment-based adapter/docking tag module (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 a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat. Biotechnol. 1486-1492, 2003).

In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first of a pair of affinity domains and the second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a pair of a second domain of affinity domains, wherein the first domain of the pair of affinity domains and the second domain of the pair of affinity domains are less than 1×10 ⁻⁷ M, less than 1×10 ⁻⁸ M; with a dissociation equilibrium constant (K _D ) of less than 1×10 ⁻⁹ M, less than 1×10 ⁻¹⁰ M, less than 1×10 ⁻¹¹ M, less than 1×10 ⁻¹² M, or less than 1×10 ⁻¹³ M Join. In some embodiments, the first domain of the pair of affinity domains and the second domain of the pair of affinity domains are about 1×10 ⁻⁴ M to about 1×10 ⁻⁶ M, about 1×10 ⁻⁵ M to about 1×10 ⁻⁷ M, about 1×10 ⁻⁶ M to about 1×10 ⁻⁸ M, about 1×10 ⁻⁷ M to about 1×10 ⁻⁹ M, about 1×10 ⁻⁸ M to about 1×10 ⁻¹⁰ M, about 1×10 ⁻⁹ M to about 1×10 ⁻¹¹ M, about 1×10 ⁻¹⁰ M to about 1×10 ⁻¹² M, about 1×10 ⁻¹¹ M to About 1×10 ⁻¹³ M, about 1×10 ⁻⁴ M to about 1×10 ⁻⁵ M, about 1×10 ⁻⁵ M to about 1×10 −6 M, about 1× ^{10 −6 M} to about 1 ×10 ⁻⁷ M, about 1×10 ⁻⁷ M to about 1×10 ⁻⁸ M, about 1×10 ⁻⁸ M to about 1×10 ^{−9 M, about 1×10 −9 M} to about 1×10 ⁻¹⁰ M, about 1×10 ⁻¹⁰ M to about 1×10 ⁻¹¹ M, about 1×10 ⁻¹¹ M to about 1×10 ⁻¹² M, or about 1×10 ⁻¹² M to about 1×10 ⁻ They bind to each other with a K _D of ¹³ M (including these upper and lower limits). Determine _{the KD} value for binding of the first domain of the pair of affinity domains and the second domain of the pair of affinity domains using any of a variety of different methods known in the art (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, biomolecule binding kinetic assays, etc.).

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

In some embodiments, any of the first and/or second domains of a pair of affinity domains disclosed herein is the first and/or second domain of the pair of affinity domains One or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids). For example, the sushi domain from the human IL-15 receptor alpha chain (IL-15Rα) has one or more additional amino acids at the N-terminus and/or C-terminus while still retaining the ability to bind soluble IL-15. can include Additionally or alternatively, soluble IL-15 may have at its N-terminus and/or C-terminus one or more Additional amino acids can be included.

Non-limiting examples of sushi domains from the alpha chain of IL-15 receptor alpha (IL-15Rα) are at least 70% identical, at least 75% identical, at least 80% identical, at least 85 It can include sequences that are % identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical. In some embodiments, the sushi domain from the IL-15Rα alpha chain is ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATA AGGCTACCAACGTGGCTCACTGGACAACACCCTTTTTAAAGTGCATCCGG (SEQ ID NO: 114).

In some embodiments, the soluble IL-15 is at least 70% identical to, at least 7 5% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, It can include sequences that are at least 99% identical, or 100% identical. In some embodiments, the soluble IL-15 is AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGA GAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATAC It can be encoded by a nucleic acid comprising the sequence of CTCC (SEQ ID NO: 116).

Signal Sequence In some embodiments, the single-chain chimeric polypeptide includes a signal sequence at its N-terminus. In some embodiments, a multichain chimeric polypeptide comprises a first chimeric polypeptide that includes a signal sequence at its N-terminus. In some embodiments, the multichain chimeric polypeptide comprises a second chimeric polypeptide that includes a signal sequence at its N-terminus. In some embodiments, both the first chimeric polypeptide of the multi-chain chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide comprise a signal sequence. As understood by those of skill in the art, a signal sequence is an amino acid sequence present at the N-terminus of some endogenously produced proteins that directs the protein into the secretory pathway (e.g., proteins are produced in certain cells located in an internal organelle, located in the cell membrane, or directed to be secreted from the cell). Signal sequences are heterogeneous and differ greatly in their primary amino acid sequences. However, signal sequences are typically 16-30 amino acids in length and comprise a hydrophilic, usually positively charged, N-terminal region, a central hydrophobic domain, and a C-terminal region containing a cleavage site for the signal peptidase. include.

In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide, the second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide has the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 117). In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide, the second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide comprises the nucleic acid sequence (SEQ ID NO: 118), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO: 119), or ATGAAATGGGTGGACCTTTTATTTCTTTACTGTTCCTCTTAGCAGCGCCTACTCC (SEQ ID NO: 120).

In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide, the second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide has the 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 has the amino acid sequence MGQIVTMFEALPHIIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG ( including a signal sequence having SEQ ID NO: 122). In some embodiments, the first chimeric polypeptide of a multi-chain chimeric polypeptide, the second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, or the single-chain chimeric polypeptide has the amino acid sequence MPNHQSGSPTGSSDLLLLSGKKQRPHLALRRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLT LFLALLSVLGPPVTG( including a signal sequence having 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 has the amino acid sequence MDSKGSSQKGSRLLLLLVVSSNLLLCQGVVS ( SEQ ID NO: 124). Those skilled in the art will appreciate other signal sequences suitable for use in the first and/or second chimeric polypeptides of the multi-chain chimeric polypeptides described herein, or single-chain chimeric polypeptides. would be aware of

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 is about 10 to It contains a signal sequence that is 100 amino acids long. For example, the signal sequence is about 10-100 amino acids long, about 15-100 amino acids long, about 20-100 amino acids long, about 25-100 amino acids long, about 30-100 amino acids long, about 35-100 amino acids long, about 40 ~100 amino acids long, about 45-100 amino acids long, about 50-100 amino acids long, about 55-100 amino acids long, about 60-100 amino acids long, about 65-100 amino acids long, about 70-100 amino acids long, about 75- 100 amino acids long, about 80-100 amino acids long, about 85-100 amino acids long, about 90-100 amino acids long, about 95-100 amino acids long, about 10-95 amino acids long, about 10-90 amino acids long, about 10-85 Amino acid length, about 10-80 amino acids, about 10-75 amino acids, about 10-70 amino acids, about 10-65 amino acids, about 10-60 amino acids, about 10-55 amino acids, about 10-50 amino acids long, about 10-45 amino acids long, about 10-40 amino acids long, about 10-35 amino acids long, about 10-30 amino acids long, about 10-25 amino acids long, about 10-20 amino acids long, about 10-15 amino acids long , about 20-30 amino acids long, about 30-40 amino acids long, about 40-50 amino acids long, about 50-60 amino acids long, about 60-70 amino acids long, about 70-80 amino acids long, about 80-90 amino acids long, It can be about 90-100 amino acids long, about 20-90 amino acids long, about 30-80 amino acids long, about 40-70 amino acids long, about 50-60 amino acids long, 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 acid length.

In some embodiments, any of the signal sequences disclosed herein have one or more additional amino acids (eg, 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids). For example, a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 125) can be used in the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both or one One or more additional amino acids can be included at the N-terminus or C-terminus while still retaining the ability to direct the chain chimeric polypeptide into the secretory pathway.

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

Peptide Tags In some embodiments, the single-chain chimeric polypeptides comprise a peptide tag (eg, at the N-terminus or C-terminus of the chimeric polypeptide). In some embodiments, a multichain chimeric polypeptide comprises a first chimeric polypeptide comprising a peptide tag (eg, at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, a multichain chimeric polypeptide comprises a second chimeric polypeptide comprising a peptide tag (eg, at the N-terminus or C-terminus of the second chimeric polypeptide). In some embodiments, both the first chimeric polypeptide of the multi-chain chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide 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 is two or more of peptide tags.

Exemplary peptide tags that may be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, or a single-chain chimeric polypeptide include: AviTag (GLNDIFEAQKIEWHE, SEQ. 130), HA tag, hemagglutinin-derived peptide (YPYDVPDYA, SEQ ID NO: 131), his tag (HHHHH (SEQ ID NO: 132), HHHHHH (SEQ ID NO: 133), HHHHHHH (SEQ ID NO: 134), HHHHHHHH (SEQ ID NO: 135), HHHHHHHHH ( SEQ ID NO: 136), or HHHHHHHHHH (SEQ ID NO: 137)), myc tag (EQKLISEEDL, SEQ ID NO: 138), NE tag (TKENPRSNQEESYDDNES, SEQ ID NO: 139), S tag (KETAAAKFERQHMDS, SEQ ID NO: 140), SBP tag (MDEKTTGWRGGHVVEGLA GELEQLRARLEHHPQGQREP, sequence No. 141), Softag1 (SLAELLNAGLGGS, SEQ ID NO: 142), Softag3 (TQDPSRVG, SEQ ID NO: 143), Spot tag (PDRVRAVSHWSS, SEQ ID NO: 144), Strep tag (WSHPQFEK, SEQ ID NO: 145), TC tag (CCPGCC, SEQ ID NO: 146 ), Ty tag (EVHTNQDPLD, SEQ ID NO: 147), V5 tag (GKPIPNPLLGLDST, SEQ ID NO: 148), VSV tag (YTDIEMNRLGK, SEQ ID NO: 149), and Xpress tag (DLYDDDDK, SEQ ID NO: 150). not. In some embodiments, the tissue factor protein is a peptide tag.

A peptide tag that may be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, or a single-chain chimeric polypeptide, is each a multi-chain Any of a variety of uses involving chimeric polypeptides or single-chain chimeric polypeptides can be used. For example, peptide tags can be used for purification of multi-chain chimeric polypeptides or single-chain chimeric polypeptides. As one non-limiting example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of a multi-chain chimeric polypeptide ( e.g., a recombinantly expressed second chimeric polypeptide), or both, or a single chain chimeric polypeptide, may comprise a myc tag, the myc tagged first chimeric polypeptide, the myc tagged second chimeric polypeptide Polypeptides, or both, or multiple-chain chimeric polypeptides, including single-chain chimeric polypeptides, can be purified using an antibody that recognizes the myc-tag. One non-limiting example of an antibody that recognizes the myc tag is 9E10 available from the non-commercial Development Studies Hybridoma Bank. As another non-limiting example, a first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), a second chimeric polypeptide of a multi-chain chimeric polypeptide ( e.g., a recombinantly expressed second chimeric polypeptide), or both, or a single chain chimeric polypeptide can include a histidine tag, a histidine-tagged first chimeric polypeptide, a histidine-tagged second chimeric polypeptide, Polypeptides, or both, or multi-chain chimeric polypeptides, including histidine-tagged single-chain chimeric polypeptides, can be purified using nickel or cobalt chelates. Those skilled in the art will recognize other suitable tags and agents to bind to those tags for use in purifying single-chain or multi-chain chimeric polypeptides. In some embodiments, the peptide tag is removed from the first chimeric polypeptide and/or the second chimeric polypeptide of a multi-chain chimeric polypeptide or the single-chain chimeric polypeptide after purification. In some embodiments, the peptide tag is not removed from the first chimeric polypeptide and/or the second chimeric polypeptide of a multi-chain chimeric polypeptide, or the single-chain chimeric polypeptide after purification.

A peptide tag that may be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, or a single-chain chimeric polypeptide may each be, for example, Immunoprecipitation of multi-chain chimeric polypeptides or single-chain chimeric polypeptides, imaging of multi-chain chimeric polypeptides or single-chain chimeric polypeptides (e.g., by Western blotting, ELISA, flow cytometry, and/or immunocytochemistry) ), and/or for solubilization of multi-chain chimeric polypeptides or single-chain chimeric polypeptides, 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 is about 10 to It contains a peptide tag that is 100 amino acids long. For example, peptide tags are about 10-100 amino acids long, about 15-100 amino acids long, about 20-100 amino acids long, about 25-100 amino acids long, about 30-100 amino acids long, about 35-100 amino acids long, about 40 ~100 amino acids long, about 45-100 amino acids long, about 50-100 amino acids long, about 55-100 amino acids long, about 60-100 amino acids long, about 65-100 amino acids long, about 70-100 amino acids long, about 75- 100 amino acids long, about 80-100 amino acids long, about 85-100 amino acids long, about 90-100 amino acids long, about 95-100 amino acids long, about 10-95 amino acids long, about 10-90 amino acids long, about 10-85 Amino acid length, about 10-80 amino acids, about 10-75 amino acids, about 10-70 amino acids, about 10-65 amino acids, about 10-60 amino acids, about 10-55 amino acids, about 10-50 amino acids long, about 10-45 amino acids long, about 10-40 amino acids long, about 10-35 amino acids long, about 10-30 amino acids long, about 10-25 amino acids long, about 10-20 amino acids long, about 10-15 amino acids long , about 20-30 amino acids long, about 30-40 amino acids long, about 40-50 amino acids long, about 50-60 amino acids long, about 60-70 amino acids long, about 70-80 amino acids long, about 80-90 amino acids long, It can be about 90-100 amino acids long, about 20-90 amino acids long, about 30-80 amino acids long, about 40-70 amino acids long, about 50-60 amino acids long, or any range therebetween. In some embodiments, the peptide tag has about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 Amino acid length.

The peptide tag contained in 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 can be of any suitable length. can be as small as For example, peptide tags can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids long. In embodiments in which the single-chain or multi-chain chimeric polypeptide comprises more than one peptide tag, the two or more peptide tags can be of the same length or different lengths. In some embodiments, any of the peptide tags disclosed herein include one or more additional peptides at the N-terminus and/or C-terminus, so long as the function of the peptide tag remains intact. It may contain amino acids (eg, 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids). For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 138) has one Additional amino acids above may be included.

Exemplary Embodiments of Single Chain Chimeric Polypeptides—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 are independently CD3 (e.g., human CD3) or CD28 (eg, human CD28). In some embodiments, the first target binding domain specifically binds CD3 (eg, human CD3) and the second target binding domain specifically binds CD28 (eg, human CD28). In some embodiments, the first target binding domain specifically binds CD28 (eg, human CD28) and the second target binding domain specifically binds CD3 (eg, human CD3).

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 polypeptide has a linker sequence between the first target binding domain and the soluble tissue factor domain (e.g., any of the exemplary linkers).

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 polypeptide has a linker sequence (e.g., as described herein) between the soluble tissue factor domain and the second target binding domain. any of the exemplary linkers).

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., exemplary antigen binding domains described herein). either). In some embodiments of these single chain chimeric polypeptides, the antigen binding domain comprises a scFv or single domain antibody.

A non-limiting example of a scFv that specifically binds to CD3 is
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGGSGGGGGSQVQLQQSGAELARPGASVK at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 8 8% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the scFv that specifically binds to CD3 is
CAGATCGTGCTGACCCAAAGCCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCG CTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGAT CCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCCGGTCAAGGTTTAGAGTGGATCGGATATAT CAACCCTTCCCGGGGGCTACACCAACTATAACCAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTACTACTGCGCTAGGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGGTACC ACTTTAACCGTCAGCAGC (SEQ ID NO: 152), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Non-limiting examples of scFv that specifically bind CD28 are:
VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGGSGGGGSGGGGSDIEMTQSPAIM SASLGERVTMTCTASSVSSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (SEQ ID NO: 153), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the scFv that specifically binds to CD28 is
GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTTCACCTCCTATGTGATCCAGTGGGTCAAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCA AATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGA GGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTG TGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGGATGCCGCCACCTACTTTTTGTCACCAGTACCCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAA AGAGG (SEQ ID NO: 154), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these single-chain chimeric polypeptides, the first target binding domain and/or the second target binding domain are soluble receptors (eg, soluble CD28 receptor or soluble CD3 receptor). be. In some embodiments of these single-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein.

In some embodiments, the single-chain chimeric polypeptide comprises
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGGSGGGGGSQVQLQQSGAELARPGASVK MSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKS GDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNNTNEFLIDVDKGEN YCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTT at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
CAGATCGTGCTGACCCAAAGCCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCG CTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCCTTCACATTCGGATCTGGCACCAAGCTCGAAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGTGGAT CCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTTACTCGTTACACAATGCATTGGGTCAAGCAGAGGCCCCGGTCAAGGTTTAGAGTGGATCGGATATAT CAACCCTTCCCGGGGGCTACACCAACTATAACCAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTACTACTGCGCTAGGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGGTACC ACTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAAATCCGGAGACTGGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGT GATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTTCCTACCCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCTACCATCCAGAGCTTCGAGCAAG TTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAAGACCGCTAAGACCAACACCAACGAGTTTTT ATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGG TGCTTCCGTGAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTTAACCTCCGACAA AAGCTCCATCACAGCCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGCGGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGAC ATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCGGAAGCTCCCCTAAACTGTGCCATCTACAGCACCAGCAATCTCGCCAGCGGCGTGCCCCCTA at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical , at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
MKWVTFISLLFLFSSAYSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSSYSLTISGMEAEDAATYYCQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGG SQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTTDKSSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGTTNTVAAYNLTWKSTNFKTILE WEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLIYTLYYWKSSSSG KKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREVQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSLTSEDSAL YYCARWGDGNYWGRGTTLTVSSGGGGSGGGGGSGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (SEQ ID NO: 15 7), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTATTCAGCTCCGCCTATTCCCAGATCGTGCTGACCCAAAGCCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAAC CAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCCTGAAGACGCTGCCACCTACTATTGCCAGCCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGC ACCAAGCTCGAAAATCAATCGTGGAGGAGGTGGCAGCGGCGGCGGGTGGATCCGGCGGAGGAGGAAGCCAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTTACACAATG CATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGGCTACACCAACTATAACCAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGGAGGACTCCGCTGTTACT ACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTTAACCGTCAGCAGCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAG ATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTTCACACAACAGACACCGAGTGTGATTTAACCGACGAAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCTCTCTACGAGAATTCCCCCGA ATTCACCCTTATTTAGAGACCAATTTAGGCCAGCTCTACCATCCAGAGCTTCGAGCAAGTTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTG GAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGGC GAGTTCCGGGAGGTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTA CAACGACTATACCAAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCCGGTGGGGGCGACGGCCAATTACTGGGGACGGGGGCCACAACACTGACCGTGAGCAGC GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCCGACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTCTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCT CCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGGCGTGCCCCCTAGAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAA ACTGGAGACAAAGAGG (SEQ ID NO: 158), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Embodiments of Single Chain Chimeric Polypeptides—Type B
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 are independently IL-2 receptor (eg, 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 polypeptide has a linker sequence between the first target binding domain and the soluble tissue factor domain (e.g., any of the exemplary linkers).

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 polypeptide has a linker sequence (e.g., a linker sequence described herein) between the soluble tissue factor domain and the second target binding domain. any of the exemplary linkers).

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. A non-limiting example of an IL-2 protein that specifically binds to the IL-2 receptor is
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 78), at least 80% identical to (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the IL-2 protein that specifically binds to the IL-2 receptor is
GCACCTACTTCAAGTTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGA AGAACTCAAAACCTCTGGAGGAAGTGCTAAAATTTAGCTACAAAGCAAAACTTTCACTTAAGACCCAGGGGACTTAATCAGCAATATCAACGTAATAGTTTCTGGAACTAAAGGGATCTGAAAACAATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCT at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the IL-2 protein that specifically binds to the IL-2 receptor is
GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCCAAGCTGACTCGTATGCTGACCTTCAAGTTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGA GCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGG At least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

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

In some embodiments, the single-chain chimeric polypeptide comprises
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSGTTNTVAAYNLTW KSTN FKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLIYTLY YWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRP at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCCAAGCTGACTCGTATGCTGACCTTCAAGTTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGA GCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGG ATCACCTTCTGCCAGTCCATCATCTCCACTTTAACCAGCGGCCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTT TCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCA CCATCCAAAGCTTTGAGCAAGTTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAA AACCAACACAAACGAGTTTTTTAATCGACGTGGATAAAGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGGAGTTCCGGGAGGCACCTACTTCAAGTTCT ACAAAGAAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAA GTGCTAAATTTAGCTCAAAGCAAAACTTTTCACTTAAGACCCAGGGGACTTAATCAGCAATATCAACGTAATAGGTTCTGGAACTAAAGGGGATCTGAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCT CAACACTAACT (SEQ ID NO: 162), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
MKWVTFISLLFLFSSSAYSAPTSSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIIST LTSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFREAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPL at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCGCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGCATCAACAACTACAGAACCCCAAGCTGACTCGTATGCTGACCTTCAA GTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCAT GTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTTAACCAGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCA AGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGC GAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAA GATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAG TGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGGCACCTACTTCAAGTTTCTACAAAAGAAAAACACGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAGAATCCCAAACTCACCAGGATGCTCACATTTTAAGTTTTTACATGCCCAAGA AGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAAATTTAGCTCAAAGCAAAACTTTTCACTTAAGACCCAGGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGA CAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT (SEQ ID NO: 164), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Embodiments of Single Chain Chimeric Polypeptides—Type C
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 are independently IL-15 receptor (eg, 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 polypeptide has a linker sequence between the first target binding domain and the soluble tissue factor domain (e.g., any of the exemplary linkers).

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 polypeptide has a linker sequence (e.g., a linker sequence described herein) between the soluble tissue factor domain and the second target binding domain. any of the exemplary linkers).

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 protein. A non-limiting example of an IL-15 protein that specifically binds to the IL-15 receptor is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 84% identical, at least 8 6% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the IL-15 protein that specifically binds to the IL-15 receptor is
AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCCACTCTGTACACTGAGAGCGACGTGCACCCTAGGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGCGATGCCAG CATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGC (SEQ ID NO: 165), at least 80% identical to (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical to , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the IL-15 protein that specifically binds to the IL-15 receptor is
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 166), and at least 8 0% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

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

In some embodiments, the single-chain chimeric polypeptide comprises
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQV YTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNE FLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNNVTESGCKECEELEEKNIKEFLQSFVHIV QMFINTS (SEQ ID NO: 167), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCCACTCTGTACACTGAGAGCGACGTGCACCCTAGGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGCGATGCCAG CATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGCAGCGGCACAACCA ACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAG ATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTTGAGCAAGTTGGCACAAAGGTGAATGTGA CAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTTAATCGACGTGGATAAAGGCGGAA AACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATAT CGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGT at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
MKWVTFISLLFLFSSAYSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGTTNTVAAYNLTWK STNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLIYTLYY WKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEEL EEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 169), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the single-chain chimeric polypeptide comprises
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCAACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGTGCACCCTAGCTGCAAGGTGACTGCCAT GAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCCAGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGGAGAAGAACATCAAGGAGTTTT TACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTTAGCAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAG TCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCGCCGGCCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATAGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAAT TTAGGACAGCCCCACCATCCAAAGCTTTGAGCAAGTTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAA GAAGACAGCTAAACCAACACAAACGAGTTTTTTAATCGACGTGGATAAAGGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCGGGAGAACTGG GTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGT at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92 % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type A
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently IL-18 receptor or IL It specifically binds to -12 receptors. In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of 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 are agonist antigen binding domains. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain are each agonist antigen binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises a scFv or single domain antibody.

In some embodiments of these multichain 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 multichain 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 multichain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to IL-18 receptor or IL-12 receptor. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-12 receptor and the second target binding domain specifically binds the IL-18 receptor. Join. In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-18 receptor and the second target binding domain specifically binds the IL-12 receptor. Join.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain comprises soluble IL-18 (eg, soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILLKEDELGDRSIMFT VQNED (SEQ ID NO: 109), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is
TACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCCGGGGCAT GGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCTTTAAGGAAATGAACCCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTAC GAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGGATCGTTCCATCATGTTCACCGTCCAAACGAGGAT (SEQ ID NO: 171), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain comprises soluble IL-12 (eg, soluble human IL-12). In some embodiments of these multichain chimeric polypeptides, the soluble human IL-15 comprises a soluble human IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence. In some embodiments of these multichain chimeric polypeptides, the soluble IL-15 human IL-15 has a linker sequence ( For example, any of the exemplary linker sequences described herein). In some examples of these multichain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12β (p40) sequence is
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCS (SEQ ID NO: 81), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human IL-12β (p40) is
ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGAACCCTCACAATCCAAGTTAA GGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGGAGGCGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAAAACTACAGCGGG TCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAGCAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGCGATACAAGGAATACGAGTACAGCGTGGAGTGCCAAGA AGATAGCGCTTGTCCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAAACTCAAGTACGAGAACTACACCCTCCTCCTTCTTATCCGGGACATCATTAAGCCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAATAGCCGGCAAGTTGAGGTCT CTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCTCCATCAGCGTGAGGGCTCAAGATC GTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (SEQ ID NO: 172), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12α (p35) is
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSSIYEDLKMYQVEFKTMNAKLMDPKRQIFLDQNMLAAVIDELMQALN FNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 80), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12α (p35) is
CGTAACCTCCCCGTGGCTACCCCCGATCCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAAGATGTACCAAGTGGAGTTCAAGACCCATGAACGCCAA GCTGCTCATGGACCCTAAACGGCAGATCTTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTACTCCACGCCTTTAGGATCCG GGCCGTGACCATTGACCGGGTCATGAGCTATTAAACGCCAGC (SEQ ID NO: 173), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILLKEDELGDRSIMFT VQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVE at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
TACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCCGGGGCAT GGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTAC GAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGGATTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGGATCGTTCCATCATGTTCACCGTCCAAACGAGGATAGCGGCCACAACCAACACAGTCGCGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCGGCCA ATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGACAACACCTTTCTCA GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAAT AGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCCACTTTATACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACC GCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCACGGCCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTT TCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 175), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSSDIIFQRSVPGHDNKMQFESSSYEGYFLACEKERD LFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQV GTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCF LLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 176), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTCACATTTATCCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGCTACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCCGGGA CAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCA GCGGTCCGTGCCCGGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGCGATCGTTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGG CACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCCGACACCGAGTGCGATCTCACCGATGAGATCGT GAAAGATGTGAAACAGACCTACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCCACCATCCAAAGCTTTTGAGCAAGTTTGGCACAAAGGTGA ATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAG GCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCAT GCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGA CAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 177), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCSGGGGSGGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSSIYEDLKMYQV EFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 178 ), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical to % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGAACCCTCACAATCCAAGTTAA GGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGGAGGCGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAAAACTACAGCGGG TCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAGCAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGCGATACAAGGAATACGAGTACAGCGTGGAGTGCCAAGA AGATAGCGCTTGTCCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAAACTCAAGTACGAGAACTACACCCTCCTCCTTCTTATCCGGGACATCATTAAGCCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAATAGCCGGCAAGTTGAGGTCT CTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCTCCATCAGCGTGAGGGCTCAAGATC GTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCCGATCCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGGA GCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCCTCAACTCTCGTGAAAACCAGCTTCATCACAAATGGCTCTTGT TTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAA ACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAAACTGTGCATTTTTACTCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTAAACGCCAGCATTACATGCCCCCTCCCCATGAGCGTGGAGCACGCC GACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTAAAGTGCATCCGG (SEQ ID NO: 179), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWW LTTISTDLTFSVKSSRGSSDPQGVTCGAATTLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGGSGGGGSGGGGSRNLPPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSF MMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRRYICNSGFKRKAGTSSLTECVLNKATNV AHWTTPSLKCIR (SEQ ID NO: 180), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCCATTTGGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAAATGGTGGTGCTCACTTGTGACACCCCCCGAAGAAGACGGCATCACTTGGACCCT CGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTTATTATTATTACACAGAAGGAAGACGGGAATCTGGGTCCACCGACATTTTTAAAGATCAGAAG GAGCCCAAGAATAAGACCTTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGGTCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCCTCAGCGCTGAGA GGGTTCGTGGCGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCCGATC CTCCTAAGAATTTACAGCTGAAGCCTCTCAAAATAGCCGGCAAGTTGAGGTCTCTTGGGGAATATCCCGACACTTGGAGCACACCCCCACAGCCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGC GCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCC GATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA GAACGAGTCTTGTCTCAACTCTCGTGAAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAG ATCTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTTAGGATCCGGGCCGTGACCATTGACCGGGTCAT GAGCTATTTAAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGATAAGGCTACCAACGTG GCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 181), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type B
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently IL-21 receptor or TGF. - binds specifically to β; In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, one or both of the first and second target binding domains are soluble IL-21 (eg, soluble human IL-21 polypeptide) or A soluble TGF-β receptor (eg, a soluble TGFRβRII receptor). In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently a soluble IL-21 or a soluble TGF-β receptor (eg, soluble TGFRβRII). receptor). In some embodiments of these multichain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the IL-21 receptor or TGF-β. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds IL-21 receptor and the second target binding domain specifically binds TGF-β. . In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds TGF-β and the second target binding domain specifically binds IL-21 receptor. .

In some embodiments of these multichain chimeric polypeptides, the first target binding domain comprises soluble IL-21 (eg, soluble human IL-21). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain comprises a soluble TGF-β receptor (eg, a soluble TGFRβRII receptor (eg, a soluble human TGFRβRII receptor)). In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII receptor is
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the human TGFβRII receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTW KSTN FKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLIYTLY YWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 189), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGT GCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTTACGAGAATTCCCCCCGAATTCACCCCTTATTTAGAGAGACCAATTTAGGCCA GCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGGAATAACACATTTTTATCCCTCCGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAGACCG CTAAGACCAACACCAACGAGTTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCCTTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCAT CAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATC ATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 190), at least 80% identical (e.g., at least 82% identical, at least 84 % identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92 % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNT FLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENL at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGA CCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTAC GAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCACGGCTCCGAGGACTCCTCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCCAAGC CCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGTCTTTTTCTCACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCT GGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAAGGACTTTAGTGCGCGCGGAATAACACATTTTTATCCCCTCCGGGATGGTGTTCGGCA AAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAGACCGCTAAGACCAACACCAACGAGTTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTTGAG TGCATGGGCCAAGAAAGGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTG CAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTTGTGCACATTGTCC AGATGTTCATCAATACCTCC (SEQ ID NO: 192), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 193), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATCACGTGTCCTCCTCCTATGTCCG TGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGGAGTGCGTGTTGAACAAGGCCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA (sequence No. 194), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADI at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCCGACATCACGTGTCCTCCCTCCTAGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type C
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently IL-7 receptor or IL It specifically binds to the -21 receptor. In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, one or both of the first and second target binding domains are soluble IL-21 (eg, soluble human IL-21 polypeptide) or Soluble IL-7 (eg, soluble human IL-7 polypeptide). In some embodiments of these multichain 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 multichain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to IL-21 receptor or IL-7 receptor. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-21 receptor and the second target binding domain specifically binds the IL-7 receptor. Join. In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-7 receptor and the second target binding domain specifically binds the IL-21 receptor. Join.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain comprises soluble IL-21 (eg, soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCA AATACAGGAAACAATGAAAAGGATAATCAATGTATCAATTAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAGATGATTCATCA at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 sequence is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEH (SEQ ID NO: 79), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTAT TCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAGTTTCAGAAGGCCAACAATACTGTTGAACTGCACTGGCCAGGTTAAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGA AGAAAAAAATCTTAAAGGAACAGAAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (SEQ ID NO: 198), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTW KSTN FKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLIYTLY YWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECE ELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 199), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCA AATACAGGAAACAATGAAAAGGATAATCAATGTATCAATTAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAGATGATTCATCA GCATCTGTCCCTCTAGAACACACGGAAGTGAAGATTCCTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTTGGAGTGGGAACCCAAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCA AATGCTTTTTACACAACAGACACAGGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACGGGCAGGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTTCACACCTTACCTGGAGACAAACCTC GGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTTCAGGAAGAAAAAC AGCCAAAACAAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAAGGGAATTCAGAGAAAAACTGGGTGAACGT CATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTA ATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 200), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92 % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MGVKVLFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSE DSSGTTNVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLII LANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 201), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGGGAGTGAAAGTTCTTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAA CTGTGAGTGGTCAGCTTTTTTCCTGTTTTCAGAGGCCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAAGGATAATCAATGTATCAATTAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGGAGAAGACAGAAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAA AAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGGAAGTGAAGTTCCTCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTTAATTTCAAGACAATTTTTGGAGTGGGAACCCAAAACCCG TCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACACACGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCTCTACCCGGCAGGGAATGTGGGAGAGCACCGGTTCT GCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTTCACACCTTACCTGGAGACAAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTG GCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTTAACCGGAAGAGTACAGACAGCCCG GTAGAGTGTATGGGCCAGGAGAAAAGGGGGAATTCAGAGAAAAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAG CTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTTCTGCCAATCCTTTGTGCACATTG TCCAGATGTTCATCAATACCTCC (SEQ ID NO: 202), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92 % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTAT TCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGA AGAAAAAAATCTTAAAGGAACAGAAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTTGTTGGAATAAAATTTTGATGGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTG at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94 % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MGVKVLFALICIAVAEADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLND at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 9 2% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGGGAGTGAAAGTTCTTTTTTGCCCTTATTTGTATTGCTGTGGGCCGAGGCCGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAGAAAATTGGTAGCAATTGCCTGAATAATGAATTTA ACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCCACAACAATACTGTTGAACTGCACTGGCCAGG TTAAAGGAAGAAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAAAATCTTTTAAAGGAACAGAAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTTGGAATAAAAATTTTGATGGGCACTAAAGAACACCATCAC GTGCCCTCCCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTTGTACTCCAGGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCCACTGGACAACCCCCA GTCTCAAATGCATTAGA (SEQ ID NO: 206), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type D
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently IL-7 receptor or IL It specifically binds to the -21 receptor. In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, one or both of the first and second target binding domains are soluble IL-21 (eg, soluble human IL-21 polypeptide) or Soluble IL-7 (eg, soluble human IL-7 polypeptide). In some embodiments of these multichain 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 multichain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to IL-21 receptor or IL-7 receptor. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-21 receptor and the second target binding domain specifically binds the IL-7 receptor. Join. In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-7 receptor and the second target binding domain specifically binds the IL-21 receptor. Join.

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCA AATACAGGAAACAATGAAAAGGATAATCAATGTATCAATTAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAGATGATTCATCA at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 sequence is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEH (SEQ ID NO: 79), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTAT TCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGA AGAAAAAAATCTTAAAGGAACAGAAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (SEQ ID NO: 198), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTL VRRNNTFLSLRDVFGKDLIYTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATG GGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAGATGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCCGGCAATGTGGAGA GCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGGAACAACACCTTTCTCTCGCGG GATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAA GCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCCAATC CTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLN DLCFLKRLLQEIKTCWNKILMGTKEHSGTTNVAAYNLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSF EQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCCKVTAM at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGT TCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGGCATGTTCCTGTTCAGGGCCGCCAGGAAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGG TGAAGGGCCGGAAACCTGCTGCTCTGGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCCACAACCAAC ACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGAT GTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACA GTGGAGGACGAGCGGACTTTAGTGCGGCGGCGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTTAATCGACGTGGATAAAGGCGAAA ACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATC GACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCC GGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 210), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKS at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGCACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCTCACCGAGTGCG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical to , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGA CCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTAC GAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCCTCCCATGAGCGTGGAGCACGCGACATCTGGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTAT ATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCCACCGAGTGCGTGCTGATAAGGCTACCAACGTGGCTCCACTGGACACACCCTTTTAAAAGTGCATCCGG (SEQ ID NO: 214), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% % identical, at least 92% identical, at least 94 % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type E
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently IL-18 (e.g., soluble specifically binds human IL-18) receptor, IL-12 (eg, soluble human IL-12) receptor, or CD16 (eg, anti-CD16 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 multichain chimeric polypeptides described herein, the additional target binding domain specifically binds to the CD16 or IL-12 receptor.

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional antigen binding domain is an agonist 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 agonist antigen binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises a scFv or single domain antibody.

In some embodiments of these multichain 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 multichain 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 multichain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to IL-18 receptor or IL-12 receptor. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-12 receptor and the second target binding domain specifically binds the IL-18 receptor. Join. In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-18 receptor and the second target binding domain specifically binds the IL-12 receptor. Join. In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds CD16 and the second target binding domain specifically binds the IL-18 receptor. In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds the IL-18 receptor and the second target binding domain specifically binds CD16.

In some embodiments of these multichain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains are directed to the same antigen. Binds specifically. 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 the same epitope. In some embodiments, two or more of the first target binding domain, second target binding domain, and one or more additional target binding domains comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain comprises soluble IL-18 (eg, soluble human IL-18).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILLKEDELGDRSIMFT VQNED (SEQ ID NO: 109), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-18 is
TACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCCGGGGCAT GGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCTTTAAGGAAATGAACCCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTAC GAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGGATCGTTCCATCATGTTCACCGTCCAAACGAGGAT (SEQ ID NO: 171), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain comprises soluble IL-12 (eg, soluble human IL-12). In some embodiments of these multichain chimeric polypeptides, the soluble human IL-15 comprises a soluble human IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence. In some embodiments of these multichain chimeric polypeptides, the soluble IL-15 (eg, soluble human IL-15) is a combination of a soluble IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence. Further includes a linker sequence (eg, any of the exemplary linker sequences described herein) in between. In some examples of these multichain chimeric polypeptides, the linker sequence comprises GGGGSGGGGSGGGGS (SEQ ID NO: 102).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12β (p40) sequence is
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCS (SEQ ID NO: 81), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human IL-12β (p40) is
ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGAACCCTCACAATCCAAGTTAA GGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGGAGGCGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAAAACTACAGCGGG TCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAGCAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGA AGATAGCGCTTGTCCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAAACTCAAGTACGAGAACTACACCCTCCTCCTTCTTATCCGGGACATCATTAAGCCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAATAGCCGGCAAGTTGAGGTCT CTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCTCCATCAGCGTGAGGGCTCAAGATC GTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC (SEQ ID NO: 172), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12α (p35) is
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSSIYEDLKMYQVEFKTMNAKLMDPKRQIFLDQNMLAAVIDELMQALN FNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 80), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-12α (p35) is
CGTAACCTCCCCGTGGCTACCCCCGATCCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAAGATGTACCAAGTGGAGTTCAAGACCCATGAACGCCAA GCTGCTCATGGACCCTAAACGGCAGATCTTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTACTCCACGCCTTTAGGATCCG GGCCGTGACCATTGACCGGGTCATGAGCTATTAAACGCCAGC (SEQ ID NO: 173), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the additional target binding domain comprises an scFv that specifically binds CD16 (eg, anti-CD16 scFv).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG at least 80% identical (e.g., at least 82% identical to , at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
EVQLVESGGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 217), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, heavy chain variable domains comprising sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGGAGGCAGGCTCCTGGAAAGGGCCTGGGAGTGGGTGTCCGGCATCAACTGGAACGG CGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCG TGTCCAGG (SEQ ID NO: 218), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILLKEDELGDRSIMFT VQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRR NNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVE at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
TACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCCGGGGCAT GGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTAC GAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGGATTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGGATCGTTCCATCATGTTCACCGTCCAAACGAGGATAGCGGCCACAACCAACACAGTCGCGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCAT CCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCGGCCA ATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGACAACACCTTTCTCA GCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAAT AGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCCACTTTATACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACC GCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCACGGCCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTT TCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 175), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFQRSVPGHDNKMQFESSSYEGYFLACEKERD LFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQV GTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCF at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTCACATTTATCCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGCTACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCCGGGA CAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTTGTGAGAACAAGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCA GCGGTCCGTGCCCGGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGCGATCGTTTCCATCATGTTCACCGTCCAAAACGAGGATAGCGG CACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCCGACACCGAGTGCGATCTCACCGATGAGATCGT GAAAGATGTGAAACAGACCTACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCCACCATCCAAAGCTTTTGAGCAAGTTTGGCACAAAGGTGA ATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAG GCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCAT GCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGA CAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 177), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCSGGGGSGGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSSIYEDLKMYQV EFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPA VSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGGNHVVFGGGTKLTVGHGGGGGSGGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCAASG FTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 223), at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGAACCCTCACAATCCAAGTTAA GGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGGAGGCGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAAAACTACAGCGGG TCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAGCAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGCGATACAAGGAATACGAGTACAGCGTGGAGTGCCAAGA AGATAGCGCTTGTCCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAAACTCAAGTACGAGAACTACACCCTCCTCCTTCTTATCCGGGACATCATTAAGCCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAATAGCCGGCAAGTTGAGGTCT CTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCTCCATCAGCGTGAGGGCTCAAGATC GTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCCGATCCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGGA GCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCCTCAACTCTCGTGAAAACCAGCTTCATCACAAATGGCTCTTGT TTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAA ACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAAACTGTGCATTTTTACTCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTAAACGCCAGCATTACATGCCCCCTCCCCATGAGCGTGGAGCACGCC GACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACACACCCCTCTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTG CTGTGTCCGTGGCTCTGGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCA ACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGGTGTTCGGCGCGCGGCACCAAGCTGACCGTGGGCCATGGCGCGGCGGCGGCTCCGGAGGCGGCGGGCAGCGGCGGAGGAGGATCC GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGGCAGGCTCCTGGAAAGGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCG GATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTG TCCAGG (SEQ ID NO: 224), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWW LTTISTDLTFSVKSSRGSSDPQGVTCGAATTLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVF TDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGGSGGGGSGGGGSRNLPPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSF MMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASITCPPPMSVEHADIWVKSYSLYSRRYICNSGFKRKAGTSSLTECVLNKATNV AHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGGSGGGGSEVQLVESG GGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 225), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86 % identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCCATTTGGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAAATGGTGGTGCTCACTTGTGACACCCCCCGAAGAAGACGGCATCACTTGGACCCT CGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTTATTATTATTACACAGAAGGAAGACGGGAATCTGGGTCCACCGACATTTTTAAAGATCAGAAG GAGCCCAAGAATAAGACCTTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGGTCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCCTCAGCGCTGAGA GGGTTCGTGGCGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCCGATC CTCCTAAGAATTTACAGCTGAAGCCTCTCAAAATAGCCGGCAAGTTGAGGTCTCTTGGGGAATATCCCGACACTTGGAGCACACCCCCACAGCCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGC GCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCCGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGCGGCGGAGGATCTCGTAACCTCCCCGTGGCTACCCCCC GATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAA GAACGAGTCTTGTCTCAACTCTCGTGAAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAG ATCTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTTAGGATCCGGGCCGTGACCATTGACCGGGTCAT GAGCTATTTAAAACGCCAGCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGATAAGGCTACCAACGTG GCTCACTGGACAACACCCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGG CAAGAACAACAGGCCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGGACGAGGCTGACTACTGCCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGGTTCGGCGGCGGCGGCACCAAGCTGACCGTGGGCCAT GGCGGCGGCGGCTCCGGAGGCGCGCGGCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCCTGGGTGAGGCAGG CTCCTGGAAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGG GGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG (SEQ ID NO: 226), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type F
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently IL-7 (e.g., soluble human IL-7) receptor, CD16 (eg, anti-CD16 scFv), or IL-21 (eg, soluble human IL-21) receptor. 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 multichain chimeric polypeptides described herein, the additional target binding domain specifically binds to the CD16 or IL-21 receptor.

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional antigen binding domain is an agonist 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 agonist antigen binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises a scFv or single domain antibody.

In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain specifically binds the receptor IL-7 and the second target binding domain is Binds specifically to CD16 or IL-21 receptors. In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain comprises a soluble IL-7 protein. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble IL-7 protein is soluble human IL-7. In some embodiments of any of the multichain chimeric polypeptides described herein, the second antigen binding domain comprises a target binding domain that specifically binds CD16. In some embodiments of any of the multichain chimeric polypeptides described herein, the second target binding domain comprises an scFv that specifically binds CD16. In some embodiments of any of the multichain chimeric polypeptides described herein, the second target binding domain specifically binds the IL-21 receptor. In some embodiments of any of the multichain chimeric polypeptides described herein, the second target binding domain comprises soluble IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to the IL-21 receptor. include. In some embodiments of any of the multichain chimeric polypeptides described herein, the additional target binding domain comprises soluble IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds CD16.

In some embodiments of these multichain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains are directed to the same antigen. Binds specifically. 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 the same epitope. In some embodiments, two or more of the first target binding domain, second target binding domain, and one or more additional target binding domains comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain comprises soluble IL-7 (eg, soluble human IL-7).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEH (SEQ ID NO: 79), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTAT TCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAGTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGA AGAAAAAAATCTTAAAGGAACAGAAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC (SEQ ID NO: 198), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 227), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 sequence is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCA AATACAGGAAACAATGAAAAGGATAATCAATGTATCAATTAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAGATGATTCATCA at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the additional target binding domain comprises an scFv that specifically binds CD16 (eg, anti-CD16 scFv).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG at least 80% identical (e.g., at least 82% identical to , at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
EVQLVESGGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 217), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, heavy chain variable domains comprising sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGGTGAGGCAGGCTCCTGGAAAGGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGG CGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCG TGTCCAGG (SEQ ID NO: 218), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTL VRRNNTFLSLRDVFGKDLIYTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATG GGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAGATGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCCGGCAATGTGGAGA GCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGGAACAACACCTTTCTCTCGCGG GATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAA GCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCCAATC CTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLN DLCFLKRLLQEIKTCWNKILMGTKEHSGTTNVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSF EQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCCKVTAM at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGT TCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGGCATGTTCCTGTTCAGGGCCGCCAGGAAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGG TGAAGGGCCGGAAACCTGCTGCTCTGGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCCACAACCAAC ACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGAT GTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACA GTGGAGGACGAGCGGACTTTAGTGCGGCGGCGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTTAATCGACGTGGATAAAGGCGAAA ACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATC GACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCC GGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 210), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGSGGGGSEVQLVESGGGGVVRPGG SLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 232), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG TTCTCCGGATCCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGGCGGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTGTCCGGAGGCGGCCG GCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAGGGGCCTGGAGTGGGTGT CCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGA CAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGATAAGG CTACCAACGTGGCTCACTGGACAACACCCTCTTAAAGTGCATCCGGCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTG GTCCGCCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCCACAACGCCGGCAGGAGGCAGAAGCACAGGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCA AGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCAGGCTCCGAGGACTCC (SEQ ID NO: 233), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGSGGG GSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMI HQHLSSRTHGSEDS (SEQ ID NO: 234), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCTCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAG GCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGGTTCTCCGGATCCTCCTCCGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGGC GGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCCGGAGGCGGCGGCAGCGGGCGGAGGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACG GCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACA CCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCTCCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCG GCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACACACCCTTTAAAGTGCATCCGGCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGAC CTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 235), at least 80% identical (e.g., at least 82% identical, at least 84 % identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type G
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently TGFβ (e.g., human TGFβRII receptor CD16 (eg, anti-CD16 scFv), or IL-21 (eg, soluble human IL-21) receptors. 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 multichain chimeric polypeptides described herein, the additional target binding domain specifically binds to the CD16 or IL-21 receptor.

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional antigen binding domain is an agonist 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 agonist antigen binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises a scFv or single domain antibody.

In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently TGF-β, CD16, or Binds specifically to the IL-21 receptor. In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain specifically binds TGF-β and the second target binding domain is CD16 or Binds specifically to the IL-21 receptor. In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain is a soluble TGF-beta receptor. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble TGF-β receptor is a soluble TGFβRII receptor. In some embodiments of any of the multichain chimeric polypeptides described herein, the second target binding domain specifically binds CD16. In some embodiments of any of the multichain chimeric polypeptides described herein, the second antigen binding domain comprises an antigen binding domain that specifically binds CD16. In some embodiments of any of the multichain chimeric polypeptides described herein, the second antigen binding domain comprises an scFv that specifically binds CD16. In some embodiments of any of the multichain chimeric polypeptides described herein, the second target binding domain specifically binds the IL-21 receptor. In some embodiments of any of the multichain chimeric polypeptides described herein, the second target binding domain comprises soluble IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the second target binding domain comprises soluble human IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to the IL-21 receptor. include. In some embodiments of any of the multichain chimeric polypeptides described herein, the additional target binding domain comprises soluble IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multichain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds CD16.

In some embodiments of these multichain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and one or more additional target binding domains are directed to the same antigen. Binds specifically. 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 the same epitope. In some embodiments, two or more of the first target binding domain, second target binding domain, and one or more additional target binding domains comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain comprises a TGFβRII receptor (eg, a soluble human TGFβRII receptor). In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human TGFβRII receptor is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 sequence is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCA AATACAGGAAACAATGAAAAGGATAATCAATGTATCAATTAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAGATGATTCATCA at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, light chain variable domains comprising sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG at least 80% identical (e.g., at least 82% identical to , at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
EVQLVESGGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 217), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, heavy chain variable domains comprising sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGGAGGCAGGCTCCTGGAAAGGGCCTGGGAGTGGGTGTCCGGCATCAACTGGAACGG CGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCG TGTCCAGG (SEQ ID NO: 218), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPK PVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTA KTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQ SFVHIVQMFINTS (SEQ ID NO: 236), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCC TATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTAACCACCGACACCGAGTGCGATCTCACCGATGGAGATCGTGAAAAGATGTGAAAACAGACCT ACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAG CGGACTTTAGTGCGGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDSGTTNTVAAY NLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLI YTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG CKECEELEEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCAT CCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAC CAACACAAACGAGTTTTTAATCGACGTGGATAAAGGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAG CGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATT at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical , at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGSGGGGSEVQLVESGGGGVVRPGG SLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNK ATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 232), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG TTCTCCGGATCCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGGCGGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTGTCCGGAGGCGGCCG GCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAGGGGCCTGGAGTGGGTGT CCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGA CAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGATAAGG CTACCAACGTGGCTCACTGGACAACACCCTCTTAAAGTGCATCCGGCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTG GTCCGCCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCCACAACGCCGGCAGGAGGCAGAAGCACAGGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCA AGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCAGGCTCCGAGGACTCC (SEQ ID NO: 233), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGSGGG GSEVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSRITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMI HQHLSSRTHGSEDS (SEQ ID NO: 234), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCTCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAG GCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGGTTCTCCGGATCCTCCTCCGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGGC GGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCCGGAGGCGGCGGCAGCGGGCGGAGGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACG GCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACA CCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGGATTACATGCCCCCTCCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCG GCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACACACCCTTTAAAGTGCATCCGGCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGAC CTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCA GGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 235), at least 80% identical (e.g., at least 82% identical, at least 84 % identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type H
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 independently specific for the IL-7 receptor. connect to each other. In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain each independently specifically bind to the IL-7 receptor. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise soluble IL-7 (eg, soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEH (SEQ ID NO: 79), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 227), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTL VRRNNTFLSLRDVFGKDLIYTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATG GGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAGATGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCCGGCAATGTGGAGA GCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGGAACAACACCTTTCTCTCGCGG GATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAA GCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCCAATC CTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLN DLCFLKRLLQEIKTCWNKILMGTKEHSGTTNVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSF EQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCCKVTAM at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGT TCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGGCATGTTCCTGTTCAGGGCCGCCAGGAAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGG TGAAGGGCCGGAAACCTGCTGCTCTGGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCCACAACCAAC ACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGAT GTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACA GTGGAGGACGAGCGGACTTTAGTGCGGCGGCGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTTAATCGACGTGGATAAAGGCGAAA ACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATC GACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCC GGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 210), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTAT TCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAGTTTCAGAAGGCCAACAATACTGTTGAACTGCACTGGCCAGGTTAAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGA AGAAAAAAATCTTAAAGGAACAGAAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTTGTTGGAATAAAATTTTGATGGGGCACTAAAGAACACATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTG at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94 % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLN DLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 250), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGT TCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCTGTTCAGGGCCGCCAGGAAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGG TGAAGGGCCGGAAACCTGCTGCTCTGGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATATTACATGCCCCCCT CCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTCACCAACGTGGCTCACTGGACAACACCCTCTTAAAGTGCATCC GG (SEQ ID NO: 251), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type I
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently specific for TGF-β. Join. In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain 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 multichain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind the same epitope. In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain and the second target binding domain are a soluble TGF-β receptor (eg, a soluble TGFβRII receptor, such as soluble human TGFβRII) is. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACCCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPK PVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTA KTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQ SFVHIVQMFINTS (SEQ ID NO: 236), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCC TATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTAACCACCGACACCGAGTGCGATCTCACCGATGGAGATCGTGAAAAGATGTGAAAACAGACCT ACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAG CGGACTTTAGTGCGGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDSGTTNTVAAY NLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLI YTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG CKECEELEEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCAT CCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAC CAACACAAACGAGTTTTTAATCGACGTGGATAAAGGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAG CGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATT at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical , at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 193), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCCTCCCATGAGCGTG GAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAGGCTCACCAACGTGGCTCACTGGACAACACCCTCTTAAAGTGCATCCGG (SEQ ID NO: 257) , at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADI at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAG GCTACCAACGTGGCTCACTGGACACACCCTTTAAAGTGCATCCGG (SEQ ID NO: 259), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type J
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently IL-7 receptor, IL -21 receptor, or specifically binds to the CD137L receptor. 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 multichain chimeric polypeptides described herein, the additional target binding domain is an IL-21 (eg, soluble IL-21, eg, soluble human IL-21) receptor or Binds specifically to the CD137L (eg, soluble CD137L, eg, soluble human CD137L) receptor.

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments, the second chimeric polypeptide may contain an additional target binding domain. In some embodiments, an additional target binding domain and.

In some embodiments of these multichain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional target binding domain is an agonist antigen binding domain. In some embodiments of these multichain chimeric polypeptides, the first target binding domain, the second target binding domain, and the additional target binding domain are each agonist antigen binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises a scFv or single domain antibody.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds IL-7 receptor and the second target binding domain is IL-21 receptor or CD137L receptor. binds specifically to In some embodiments, the additional target binding domain specifically binds to IL-21 receptor or CD137L receptor.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain is soluble IL-7 (eg, soluble human IL-7). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEH (SEQ ID NO: 79), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 227), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain or additional target binding domain specifically binds to the IL-21 receptor. In some embodiments of these multichain chimeric polypeptides, the second or additional target binding domain is soluble IL-21 (eg, soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain specifically binds the CD137L receptor. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to the CD137L receptor. In some embodiments of these multichain chimeric polypeptides, the second and/or additional target binding domains are soluble CD137L (eg, soluble human CD137L).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human CD137L is
REGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSSVSLALHLQPLRSAAGAAALALTVDLLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSSE (SEQ ID NO: 260), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human CD137L is
CGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCGCAGCTGGTGGCCCAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGC CTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG GCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human CD137L is
DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 262), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human CD137L is
GATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGG TGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCT CCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGGCAGCTTACCCAGGGCGCCACAGTCTTTGGACTCTTCCGGGTGACCCCCGAAAATC (SEQ ID NO: 2) 63), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTL VRRNNTFLSLRDVFGKDLIYTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATG GGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAGATGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCCGGCAATGTGGAGA GCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGGAACAACACCTTTCTCTCGCGG GATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAA GCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATC CTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLN DLCFLKRLLQEIKTCWNKILMGTKEHSGTTNVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSF EQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCCKVTAM at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGT TCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGGCATGTTCCTGTTCAGGGCCGCCAGGAAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGG TGAAGGGCCGGAAACCTGCTGCTCTGGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCCACAACCAAC ACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGAT GTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACA GTGGAGGACGAGCGGACTTTAGTGCGGCGGCGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTTAATCGACGTGGATAAAGGCGAAA ACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATC GACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCC GGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 210), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVV AGEGSGSVSLALHLQPLRSAAGAAAALALTTVDLPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE (SEQ ID NO: 268), at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCTCACCGAGTGCG TGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATG TTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCCAAGGCTGGAGTCTACTATGTCTTCTTTCAAC TAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCGCGCCTCCTCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCG at least 80% identical to CTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 269), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA at least 80% identical (e.g., at least 82% identical, at least 8 4% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGA CCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTAC GAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCCTCCCATGAGCGTGGAGCACGCGACATCTGGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTAT ATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTC CCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGG ACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGGTGGCCGGCGCGGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGAC CTGCCACCCGCCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGAC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCTCACCGAGTGCG TGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAAATGT TCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTTAGAGCTGCGGCGCGTGGTGGCCGGG CGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTTVDLPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 274), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88 % identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGA CCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTAC GAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCCTCCCATGAGCGTGGAGCACGCGACATCTGGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTAT ATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGATAAGGCCTACCAACGTGGCTCACTGGACACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGGC CTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGG CTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTC GGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 275), and at least 80% identical ( For example, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type K
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 independently IL-7 receptor or TGF. - binds specifically to β; In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds IL-7 receptor and the second target binding domain specifically binds TGF-β. . In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds TGF-β and the second target binding domain specifically binds IL-7 receptor. .

In some embodiments of these multichain chimeric polypeptides, the first target binding domain comprises a soluble IL-7 protein (eg, soluble human IL-7 protein). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 protein is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEH (SEQ ID NO: 79), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-7 is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT (SEQ ID NO: 227), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain comprises a target binding domain that specifically binds TGF-β. In some embodiments of these multichain chimeric polypeptides, the second target binding domain is a soluble TGF-β receptor (eg, soluble TGFβRII receptor, eg, soluble human TGFβRII receptor). In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACCCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTL VRRNNTFLSLRDVFGKDLIYTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHD at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATG GGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAGATGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTTAGCTACCCCGCCCGGCAATGTGGAGA GCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGGAACAACACCTTTCTCTCGCGG GATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAACCAACACAAACGAGTTTTAATCGACGTGGATAAAGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAA GCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAAT GTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCCAATC CTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 208), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLN DLCFLKRLLQEIKTCWNKILMGTKEHSGTTNVAAYNLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSF EQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCCKVTAM at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCGATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGT TCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGGCATGTTCCTGTTCAGGGCCGCCAGGAAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGG TGAAGGGCCGGAAACCTGCTGCTCTGGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGAACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCATAGCGGCCACAACCAAC ACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGAT GTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACA GTGGAGGACGAGCGGACTTTAGTGCGGCGGCGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTTAATCGACGTGGATAAAGGCGAAA ACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATC GACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCC GGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 210), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 193), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCCTCCCATGAGCGTG GAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAGGCTCACCAACGTGGCTCACTGGACAACACCCTCTTAAAGTGCATCCGG (SEQ ID NO: 257) , at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADI at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAG GCTACCAACGTGGCTCACTGGACACACCCTTTAAAGTGCATCCGG (SEQ ID NO: 259), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type L
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently TGF-β, IL-21 Binds specifically to the receptor, or CD137L receptor. 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 multichain chimeric polypeptides described herein, the additional target binding domain is an IL-21 (eg, soluble IL-21, eg, soluble human IL-21) receptor or Binds specifically to the CD137L (eg, soluble CD137L, eg, soluble human CD137L) receptor.

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the additional target binding domain is an agonist antigen binding domain. In some embodiments of these multichain chimeric polypeptides, the first target binding domain, the second target binding domain, and the additional target binding domain are each agonist antigen binding domains. In some embodiments of these multi-chain chimeric polypeptides, the antigen binding domain comprises a scFv or single domain antibody.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds TGF-β and the second target binding domain is specific for IL-21 receptor or CD137L receptor. physically connect.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain is a soluble TGF-β receptor (eg, soluble TGFβRII receptor, eg, soluble human TGFβRII receptor). In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain or additional target binding domain specifically binds to the IL-21 receptor. In some embodiments of these multichain chimeric polypeptides, the second or additional target binding domain comprises soluble IL-21 (eg, soluble human IL-21).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain or additional target binding domain specifically binds to the CD137L receptor. In some embodiments of these multichain chimeric polypeptides, the second and/or additional target binding domains comprise soluble CD137L (eg, soluble human CD137L).

In some embodiments of these multichain chimeric polypeptides, the soluble CD137L is
REGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSSVSLALHLQPLRSAAGAAALALTVDLLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSSE (SEQ ID NO: 260), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble CD137L is
CGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCGCAGCTGGTGGCCCAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGC CTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG GCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human CD137L is
DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 262), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble human CD137L is
GATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGG TGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCT CCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGGCGTCCATCTTCACACTGAGGCCAGGGCAGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTTGGACTCTTCCGGGTGACCCCCGAAAATC (SEQ ID NO: 2) 63), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPK PVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTA KTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQ SFVHIVQMFINTS (SEQ ID NO: 236), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCC TATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTAACCACCGACACCGAGTGCGATCTCACCGATGGAGATCGTGAAAAGATGTGAAAACAGACCT ACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAG CGGACTTTAGTGCGGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDSGTTNTVAAY NLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLI YTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG CKECEELEEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCAT CCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAC CAACACAAACGAGTTTTTAATCGACGTGGATAAAGGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAG CGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATT at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical , at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCTCACCGAGTGCG TGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGCGGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATG TTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCCAAGGCTGGAGTCTACTATGTCTTCTTTCAAC TAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCGCGCCTCCTCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCG at least 80% identical to CTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 269), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA at least 80% identical (e.g., at least 82% identical, at least 8 4% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGA CCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTAC GAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCCTCCCATGAGCGTGGAGCACGCGACATCTGGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTAT ATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTC CCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGG ACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGGTGGCCGGCGCGGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGAC CTGCCACCCGCCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGAC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKS YSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCTCACCGAGTGCG TGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCTGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAAATGT TCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTTAGAGCTGCGGCGCGTGGTGGCCGGG CGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGS EDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGSGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTTVDLPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 274), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88 % identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGGTGACCTTCATCAGCCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCCAGGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGA CCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTAC GAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCCATTACATGCCCCCCCTCCCATGAGCGTGGAGCACGCGACATCTGGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTAT ATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGATAAGGCCTACCAACGTGGCTCACTGGACACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGGAGGTGGCTCCGGCGGCGGAGGATCTGATCCCGCCGGGC CTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGG CTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTC GGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATC (SEQ ID NO: 275), and at least 80% identical ( For example, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type M
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 independently TGF-β or IL-21 Binds specifically to receptors. 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 multichain chimeric polypeptides described herein, the additional target binding domain is an IL-21 (eg, soluble IL-21, eg, soluble human IL-21) receptor or Binds specifically to TGF-beta receptors (eg, soluble TGF-beta receptors, eg, soluble TGFβRII receptors).

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds TGF-β and the second target binding domain is specific for TGF-β or IL-21 receptor. physically connect. In some embodiments of these multichain chimeric polypeptides, the first target binding domain is a soluble TGF-β receptor (eg, soluble TGFβRII receptor, eg, soluble human TGFβRII receptor). In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain specifically binds the IL-21 receptor. In some embodiments of these multichain chimeric polypeptides, the second target binding domain comprises soluble IL-21 (eg, human soluble IL-21). In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-21 is
at least 80% identical to QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 83), (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-21 is
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 182), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPK PVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTA KTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQ SFVHIVQMFINTS (SEQ ID NO: 236), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCC TATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTAACCACCGACACCGAGTGCGATCTCACCGATGGAGATCGTGAAAAGATGTGAAAACAGACCT ACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAG CGGACTTTAGTGCGGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDSGTTNTVAAY NLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLI YTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG CKECEELEEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCAT CCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAC CAACACAAACGAGTTTTTAATCGACGTGGATAAAGGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAG CGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATT at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical , at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIH QHLSSRTHGSEDS (SEQ ID NO: 300), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCCTCCCATGAGCGTG GAGCACGCCGACATCTGGGTGAAGAGCTATAGCCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGTAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGCAGGGGCCAG GACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAAC AACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGCAGAAGATGATCCCATCAGCACCT GTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 301), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADI WVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEK at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAG GCTACCAACGTGGCTCACTGGACAACACCCTCTTAAAGTGCATCCGGCAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGT GGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCCACAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCC AAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (SEQ ID NO: 303), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to % identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type N
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently specific for TGF-β or CD16. connect to each other. 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 multichain chimeric polypeptides described herein, the additional target binding domain is CD16 (eg, anti-CD16 scFv) or TGF-β (eg, a soluble TGF-β receptor, such as , the soluble TGFβRII receptor).

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds TGF-β and the second target binding domain specifically binds TGF-β or CD16. . In some embodiments of these multichain chimeric polypeptides, the first target binding domain is a soluble TGF-β receptor (eg, soluble TGFβRII receptor, eg, soluble human TGFβRII receptor). In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACCCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain specifically binds CD16. In some embodiments of these multichain chimeric polypeptides, the second target binding domain comprises an anti-CD16 scFv. In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, light chain variable domains comprising sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG at least 80% identical (e.g., at least 82% identical to , at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
EVQLVESGGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR (SEQ ID NO: 217), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, heavy chain variable domains comprising sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the scFv that specifically binds to CD16 is
GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGGAGGCAGGCTCCTGGAAAGGGCCTGGGAGTGGGTGTCCGGCATCAACTGGAACGG CGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCG TGTCCAGG (SEQ ID NO: 218), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPK PVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTA KTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQ SFVHIVQMFINTS (SEQ ID NO: 236), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCC TATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTAACCACCGACACCGAGTGCGATCTCACCGATGGAGATCGTGAAAAGATGTGAAAACAGACCT ACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAG CGGACTTTAGTGCGGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDSGTTNTVAAY NLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLI YTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG CKECEELEEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCAT CCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAC CAACACAAACGAGTTTTTAATCGACGTGGATAAAGGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAG CGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATT at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical , at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGGTKLTVGHGGGGG at least 80% identical to (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCCTCCCATGAGCGTG GAGCACGCCGACGACATCTGGGTGAAGAGCTATAGCCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACC CAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGGTTCTCCGGATCCT CCTCCGGCCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGCGGCTCCGGAGGCGGCGGCAGCGGCGGA GGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCTCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAGGGGCCTGGAGTGGGTGTCCGGCATCAACTG GAACGGCGGATCCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGG TGACCGTGTCCAGG (SEQ ID NO: 309), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADI WVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVV FGGGTKLTVGHGGGGGSGGGGSGGGGSEVQLVESGGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVS R (SEQ ID NO: 310), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAG GCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGTCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTG GTGATCTACGGCAAGAACAACAGGCCCTCCGGCATCCCTGACAGGTTCTCCGGATCCTCCTCGGCAACACCGCCTCCCTGACCATCACAGGGCGCTCAGGCCGGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGA CCGTGGGCCATGGCGCGCGGCGGCTCCGGAGGCGGCGGCAGCGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGG GTGAGGCAGGCTCCTGGAAAGGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTA at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type O
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each independently TGF-beta or CD137L receptor binds specifically to 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 multichain chimeric polypeptides described herein, the additional target binding domain is a TGF-β receptor (eg, a soluble TGF-β receptor, such as a soluble TGFβRII receptor) or specifically binds to the CD137L receptor.

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds TGF-β and the second target binding domain specifically binds CD137L. In some embodiments of these multichain chimeric polypeptides, the first target binding domain or additional target binding domain is a soluble TGF-β receptor (eg, a soluble TGFβRII receptor, such as a soluble human TGFβRII receptor). ).

In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, in some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCAAGCTGCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second target binding domain comprises a soluble CD137L protein (eg, soluble human CD137L protein). In some embodiments of these multichain chimeric polypeptides, the soluble human CD137L is
REGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSSVSLALHLQPLRSAAGAAALALTVDLLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSSE (SEQ ID NO: 260), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human CD137L is
CGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCGCAGCTGGTGGCCCAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGC CTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG GCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human CD137L is
DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEI (SEQ ID NO: 262), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble human CD137L is
GATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGG TGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCT CCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGGCAGCTTACCCAGGGCGCCACAGTCTTTGGACTCTTCCGGGTGACCCCCGAAAATC (SEQ ID NO: 2) 63), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPK PVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTA KTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQ SFVHIVQMFINTS (SEQ ID NO: 236), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCC TATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTAACCACCGACACCGAGTGCGATCTCACCGATGGAGATCGTGAAAAGATGTGAAAACAGACCT ACCTCGCCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAG CGGACTTTAGTGCGGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAG CGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGGAGTTCCGGGGAGAACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGAATTTAATTCAGTCCATGCATATCGACGCCACTTTATA CACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDSGTTNTVAAY NLTWKSTNFKTILEWEPKKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNNTFLSLRDVFGKDLI YTLYYWKSSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVIDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESG CKECEELEEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCGACAGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCT ATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCAT CCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAAC CAACACAAACGAGTTTTTAATCGACGTGGATAAAGGGCGAAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGGAGAACTGGGTGAACGTCATCAG CGATTTAAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATT at least 80% identical (e.g., at least 82% identical, at least 84% identical , at least 86% identical, at least 88% identical, at least 90% identical , at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSG FKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSSLALH at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical , at least 94 % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCCTCCCATGAGCGTG GAGCACCGCCGACATCTGGGTGAAGAGCTATAGCCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGTAATAAGGCTACCAACGTGGCTCCACTGGACAACACCCTCTTTAAAGTGCATCCGGGCGGTG GAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCTCGCGAGGGTTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACC CAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGCGTGGTGGCCGGCGCGGGGCTCAGGCTCCGTTTTCACTTGCGCTGCACCT GCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCC ATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA (SEQ ID NO: 317), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADI WVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLEL at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 86% identical, at least 8 8% identical, at least 90% identical, at least 92% identical, at least 94 % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACACGGCGCCGTGAAGTTTCCCCAGCTCTGCCAAGTTTCTGCGATGTCAGGGTTCAGCACCTGCGA TAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGC ATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCA GAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCT GGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCAT CTTTAGCGAGGAATACAATACCAGCAACCCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAG GCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGGGGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGCGCGGAGGATCTCGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAG CTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCCAAGGCTGGAGTCTACTATGTCTTACTTTCAACTGAGCTGCGGG CGCGTGGTGGCCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCT at least 80% identical (e.g., at least 82% identical to , at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Exemplary Multichain Chimeric Polypeptides—Type P
In some embodiments of any of the multichain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each specifically bind TGF-β. In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. A non-limiting example of this type of multi-chain chimeric polypeptide is shown in FIGS.

In some examples of these multichain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multichain chimeric polypeptides, the first chimeric polypeptide has a linker sequence (e.g., any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide is between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. Further includes a linker sequence (eg, any of the exemplary linkers described herein).

In some embodiments of these multi-chain chimeric polypeptides, the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is a combination of the second of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. Further includes a linker sequence (eg, any of the exemplary linkers described herein) in between.

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide is between the second of the pair of affinity domains and the additional target binding domain within the second chimeric polypeptide. further includes a linker sequence (eg, any of the exemplary linkers described herein).

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 within the second chimeric polypeptide. In some embodiments of these multichain chimeric polypeptides, the second chimeric polypeptide has a linker sequence ( For example, any of the exemplary linkers described herein).

In some embodiments of these multichain chimeric polypeptides, the soluble tissue factor domain can 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 pair of affinity domains described herein.

In some embodiments of these multichain chimeric polypeptides, the first target binding domain specifically binds TGF-beta and the second target binding domain specifically binds TGF-beta. . In some embodiments of these multichain chimeric polypeptides, the first target binding domain and/or the second target binding domain is a soluble TGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., a soluble human TGFβRII receptor).

In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multichain chimeric polypeptides, the soluble human TGFRβRII comprises a linker positioned between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence. In some examples of these multichain chimeric polypeptides, the linker comprises the sequence GGGGSGGGGSGGGGGS (SEQ ID NO: 102).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, sequences that are at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the first soluble human TGFRβRII receptor sequence is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 185), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the second soluble human TGFRβRII receptor sequence is
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, in some embodiments of these multichain chimeric polypeptides, the soluble TGF-β receptor is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC (SEQ ID NO: 187), and at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPK PVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTA KTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQ SFVHIVQMFINTS (SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, % identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
ATCCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAA GTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGAT GAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCCAGCT GTGCAAATTCTGCGATGTGGAGGTTTTCCACCTGCGCACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCTACCACGAT TTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGACTCAGGCACTACAAATACTGTGGCAGCATATATAATT TAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGTGTGTGACCTCACCGACGAGATTGTGAAGGATGTGGAAGCAGACGTACT TGGCACGGGTCTTCTCCTACCCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAGTGAATGTGACCGTAGAAGATGAACG GACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTTCAGGAAAGAAACAGCCAAAACAAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAAACTACTGTTTCAGTG TTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGGTGTATGGGCCAGGAGAAAAGGGGGAATTCAGAGAAAAACTGGGTGAATGTAATAAGTAATTTGAAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAA GTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAATCTGATCATCCTAGCAAACAACAGTTTTGTCTTCTAATGGGAATGTAACAGAAATCTGGATGCAAAGAAT at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide can comprise soluble IL-15 comprising a D8N amino acid substitution,
(signal peptide)
MGVKVLFALICIAVAEA
(single-chain human TGF-β receptor II homodimer)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(tissue factor)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(IL-15D8N)
NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
(SEQ ID NO: 238), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the first chimeric polypeptide is
(signal peptide)
ATGGGAGTGAAAGTTCTTTTTGCCCTTTATTGTATTGCTGTGGCCCGAGGCC
(single-chain human TGF-β receptor II homodimer)
ATCCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAA GTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGAT GAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCCAGCT GTGCAAATTCTGCGATGTGGAGGTTTTCCACCTGCGCACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCTACCACGAT TTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
TCAGGCACTACAAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCA CCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCTCTCGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGT GGGAACAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAACAGCCAAAACAAAACACTAATGAGTTTTTGATT GATGTGGATAAAGGAGAAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCCGGTAGAGTGTATGGGCCAGGAGAAAAGGGGGAATTCAGAGAA
(human IL-15D8N)
AACTGGGTGAATGTAATAAGTAATTTGAAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCA AGTATTCATGATACAGTAGAAATCTGATCATCCTAGCAAACAACAGTTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT (SEQ ID NO: 2 44), at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical to , at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSG at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCCTCCCATGAGCGTG GAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 241) , at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical to , at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is
MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGG GSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADI at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% sequences that are identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

In some embodiments, the second chimeric polypeptide is ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCCATCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGAT GTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGA GGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTG GGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGA GGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCTCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTCATGTGCTCCTGCAGCA GCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCA at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical , at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical).

Methods of Treating Age-Related Diseases or Conditions Age-related diseases or conditions (e.g., any of the exemplary types of age-related diseases or conditions described herein or known in the art) A method of treating an age-related disease or condition in a subject in need thereof, comprising: A therapeutically effective amount of one or more natural killer (NK) cell activators (e.g., as described herein or in the art) to a subject identified as having a relevant disease or condition Methods are provided herein comprising administering any of the known natural killer (NK) cell activators.

Age-related diseases or conditions in a subject in need of treatment (e.g., any of the exemplary types of age-related diseases or conditions described herein or known in the art) A method of treating a disease or condition, which age-related disease or condition (e.g., any of the exemplary types of age-related diseases or conditions described herein or known in the art) 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 A method is provided herein comprising:

Some embodiments of these methods include obtaining resting NK cells and contacting the resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators, wherein Contacting results in the generation of activated NK cells that are then administered to the subject. In some examples of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some examples of these methods, the resting NK cells are haploidentical NK cells obtained from the subject. In some examples of these methods, the resting NK cells are allogeneic resting NK cells. In some examples of these methods, the resting NK cells are induced NK cells. In some examples of any of these methods, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

In some examples of these methods, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically defined liquid culture medium. Some examples of these methods include isolating activated NK cells (and optionally administering a therapeutically effective amount of the activated NK cells to a subject, e.g., a subject described herein). further administering to any of In some embodiments of these methods, the contacting step is carried out for a period of time from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of any of the methods described herein, the age-related disease or condition is cancer, autoimmune disease, metabolic disease, neurodegenerative disease, cardiovascular disease, skin disease, It is selected from the group of progeria and frailty diseases.

Non-limiting examples of cancer include solid tumors, hematologic tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms. , multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL) ), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, Colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, 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 disease include coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

Non-limiting examples of skin disorders include wound healing, alopecia, wrinkles, senile lentigines, skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

Non-limiting examples of progeria include progeria and Hutchinson-Guilford progeria syndrome.

Non-limiting examples of frailty diseases include frailty, response to vaccination, osteoporosis, and sarcopenia.

In some embodiments of any of the age-related diseases or conditions described herein, the age-related disease or condition is age-related macular degeneration, osteoarthritis, fat wasting, chronic obstructive pulmonary disease , idiopathic pulmonary fibrosis, renal graft failure, liver fibrosis, bone loss, sarcopenia, age-related pulmonary tissue elastic loss, osteoporosis, age-related renal dysfunction, and chemically-induced renal dysfunction selected from the group of

In some embodiments of any of the age-related diseases or conditions described herein, the age-related disease or condition is type 2 diabetes or atherosclerosis.

In some embodiments of any of the methods described herein, the subject has an age-related disease or condition (e.g., one of the exemplary age-related diseases or conditions described herein) have been identified or diagnosed as having Some embodiments of any of the methods described herein use age-related diseases or conditions (e.g., any of the exemplary age-related diseases or conditions described herein). selecting subjects who have been identified or diagnosed as having

In some embodiments of these methods, administering, for example, results in a reduction (e.g., at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction in the number of senescent cells in a target tissue in a subject). , at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced , at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 10% reduced to about 99% reduced, about 10% reduced to about 95% reduced, about 10 % reduction to about 90% reduction, about 10% reduction to about 85% reduction, about 10% reduction to about 80% reduction, about 10% reduction to about 75% reduction, about 10% reduction to about 70% reduction, about 10 % reduction to about 65% reduction, about 10% reduction to about 60% reduction, about 10% reduction to about 55% reduction, about 10% reduction to about 50% reduction, about 10% reduction to about 45% reduction, about 10 % reduction to about 40% reduction, about 10% reduction to about 35% reduction, about 10% reduction to about 30% reduction, about 10% reduction to about 25% reduction, about 10% reduction to about 20% reduction, about 10 % reduction to about 15% reduction, about 15% reduction to about 99% reduction, about 15% reduction to about 95% reduction, about 15% reduction to about 90% reduction, about 15% reduction to about 85% reduction, about 15 % reduction to about 80% reduction, about 15% reduction to about 75% reduction, about 15% reduction to about 70% reduction, about 15% reduction to about 65% reduction, about 15% reduction to about 60% reduction, about 15 % reduction to about 55% reduction, about 15% reduction to about 50% reduction, about 15% reduction to about 45% reduction, about 15% reduction to about 40% reduction, about 15% reduction to about 35% reduction, about 15 % reduction to about 30% reduction, about 15% reduction to about 25% reduction, about 15% reduction to about 20% reduction, about 20% reduction to about 99% reduction, about 20% reduction to about 95% reduction, about 20 % reduction to about 90% reduction, about 20% reduction to about 85% reduction, about 20% reduction to about 80% reduction, about 20% reduction to about 75% reduction, about 20% reduction to about 70% reduction, about 20 % reduction to about 65% reduction, about 20% reduction to about 60% reduction, about 20% reduction to about 55% reduction, about 20% reduction to about 50% reduction, about 20% reduction to about 45% reduction, about 20 % reduction to about 40% reduction, about 20% reduction to about 35% reduction, about 20% reduction to about 30% reduction, about 20% reduction to about 25% reduction, about 25% reduction to about 99% reduction, about 25 % reduction to about 95% reduction, about 25% reduction to about 90% reduction, about 25% reduction to about 85% reduction, about 25% reduction to about 80% reduction, about 25% reduction to about 75% reduction, about 25 % reduction to about 70% reduction, about 25% reduction to about 65% reduction, about 25% reduction to about 60% reduction, about 25% reduction to about 55% reduction, about 25% reduction to about 50% reduction, about 25 % reduction to about 45% reduction, about 25% reduction to about 40% reduction, about 25% reduction to about 35% reduction, about 25% reduction to about 30% reduction, about 30% reduction to about 99% reduction, about 30 % reduction to about 95% reduction, about 30% reduction to about 90% reduction, about 30% reduction to about 85% reduction, about 30% reduction to about 80% reduction, about 30% reduction to about 75% reduction, about 30 % reduction to about 70% reduction, about 30% reduction to about 65% reduction, about 30% reduction to about 60% reduction, about 30% reduction to about 55% reduction, about 30% reduction to about 50% reduction, about 30 % reduction to about 45% reduction, about 30% reduction to about 40% reduction, about 30% reduction to about 35% reduction, about 35% reduction to about 99% reduction, about 35% reduction to about 95% reduction, about 35 % reduction to about 90% reduction, about 35% reduction to about 85% reduction, about 35% reduction to about 80% reduction, about 35% reduction to about 75% reduction, about 35% reduction to about 70% reduction, about 35 % reduction to about 65% reduction, about 35% reduction to about 60% reduction, about 35% reduction to about 55% reduction, about 35% reduction to about 50% reduction, about 35% reduction to about 45% reduction, about 35 % reduction to about 40% reduction, about 40% reduction to about 99% reduction, about 40% reduction to about 95% reduction, about 40% reduction to about 90% reduction, about 40% reduction to about 85% reduction, about 40 % reduction to about 80% reduction, about 40% reduction to about 75% reduction, about 40% reduction to about 70% reduction, about 40% reduction to about 65% reduction, about 40% reduction to about 60% reduction, about 40 % reduction to about 55% reduction, about 40% reduction to about 50% reduction, about 40% reduction to about 45% reduction, about 45% reduction to about 99% reduction, about 45% reduction to about 95% reduction, about 45 % reduction to about 90% reduction, about 45% reduction to about 85% reduction, about 45% reduction to about 80% reduction, about 45% reduction to about 75% reduction, about 45% reduction to about 70% reduction, about 45 % reduction to about 65% reduction, about 45% reduction to about 60% reduction, about 45% reduction to about 55% reduction, about 45% reduction to about 50% reduction, about 50% reduction to about 99% reduction, about 50 % reduction to about 95% reduction, about 50% reduction to about 90% reduction, about 50% reduction to about 85% reduction, about 50% reduction to about 80% reduction, about 50% reduction to about 75% reduction, about 50 % reduction to about 70% reduction, about 50% reduction to about 65% reduction, about 50% reduction to about 60% reduction, about 50% reduction to about 55% reduction, about 55% reduction to about 99% reduction, about 55 % reduction to about 95% reduction, about 55% reduction to about 90% reduction, about 55% reduction to about 85% reduction, about 55% reduction to about 80% reduction, about 55% reduction to about 75% reduction, about 55 % reduction to about 70% reduction, about 55% reduction to about 65% reduction, about 55% reduction to about 60% reduction, about 60% reduction to about 99% reduction, about 60% reduction to about 95% reduction, about 60 % reduction to about 90% reduction, about 60% reduction to about 85% reduction, about 60% reduction to about 80% reduction, about 60% reduction to about 75% reduction, about 60% reduction to about 70% reduction, about 60 % reduction to about 65% reduction, about 65% reduction to about 99% reduction, about 65% reduction to about 95% reduction, about 65% reduction to about 90% reduction, about 65% reduction to about 85% reduction, about 65 % reduction to about 80% reduction, about 65% reduction to about 75% reduction, about 65% reduction to about 70% reduction, about 70% reduction to about 99% reduction, about 70% reduction to about 95% reduction, about 70 % reduction to about 90% reduction, about 70% reduction to about 85% reduction, about 70% reduction to about 80% reduction, about 70% reduction to about 75% reduction, about 75% reduction to about 99% reduction, about 75 % reduction to about 95% reduction, about 75% reduction to about 90% reduction, about 75% reduction to about 85% reduction, about 75% reduction to about 80% reduction, about 80% reduction to about 99% reduction, about 80 % reduction to about 95% reduction, about 80% reduction to about 90% reduction, about 80% reduction to about 85% reduction, about 85% reduction to about 99% reduction, about 85% reduction to about 95% reduction, about 85 % reduction to about 90% reduction, about 90% reduction to about 99% reduction, about 90% reduction to about 95% reduction, or about 95% reduction to about 99% reduction in target tissue in a subject prior to treatment. (compared to the number of senescent cells in ).

In some embodiments of these methods, administering results in an increase in levels of IFN-γ, cytotoxic granule granzymes, and/or perforin in the subject (e.g., at least a 5% increase, at least a 10% increase, at least a 15% increase, at least 20% increase, at least 25% increase, at least 30% increase, at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase, at least 85% increase, at least 90% increase, at least 95% increase, or at least 99% increase, or about 10% increase to about 500% increase (or (any of the subranges of this range described herein) (compared to levels in pretreatment subjects or similar control subjects not receiving treatment).

In some embodiments, these methods can result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., one or more symptoms of cancer in a subject prior to treatment). compared to the number, severity, or frequency of symptoms). In some embodiments, these methods reduce the volume of one or more solid tumors in a subject (e.g., about 1% to about 99% reduction, about 1% to about 95% reduction, about 1% Reduced to about 90%, Reduced about 1% to about 85%, Reduced about 1% to about 80%, Reduced about 1% to about 75%, Reduced about 1% to about 70%, Reduced about 1% Reduced to about 65% reduced, about 1% reduced to about 60% reduced, about 1% reduced to about 55% reduced, about 1% reduced to about 50% reduced, about 1% reduced to about 45% reduced, about 1% Decrease to about 40% decrease, about 1% decrease to about 35% decrease, about 1% decrease to about 30% decrease, about 1% decrease to about 25% decrease, about 1% decrease to about 20% decrease, about 1% Reduced to about 15% reduced, about 1% reduced to about 10% reduced, about 1% reduced to about 5% reduced, about 5% reduced to about 99% reduced, about 5% reduced to about 95% reduced, about 5% Reduced to about 90% reduced, about 5% reduced to about 85% reduced, about 5% reduced to about 80% reduced, about 5% reduced to about 75% reduced, about 5% reduced to about 70% reduced, about 5% Reduced to about 65% reduced, about 5% reduced to about 60% reduced, about 5% reduced to about 55% reduced, about 5% reduced to about 50% reduced, about 5% reduced to about 45% reduced, about 5% Decrease to about 40% decrease, about 5% decrease to about 35% decrease, about 5% decrease to about 30% decrease, about 5% decrease to about 25% decrease, about 5% decrease to about 20% decrease, about 5% about 15% reduction, about 5% reduction to about 10% reduction, about 10% reduction to about 99% reduction, about 10% reduction to about 95% reduction, about 10% reduction to about 90% reduction, about 10% about 85% reduction, about 10% reduction to about 80% reduction, about 10% reduction to about 75% reduction, about 10% reduction to about 70% reduction, about 10% reduction to about 65% reduction, about 10% About 10% to about 55% reduction, about 10% to about 50% reduction, about 10% to about 45% reduction, about 10% to about 40% reduction, about 10% about 10% to about 30% reduction, about 10% to about 25% reduction, about 10% to about 20% reduction, about 10% to about 15% reduction, about 15% about 99% reduction, about 15% reduction to about 95% reduction, about 15% reduction to about 90% reduction, about 15% reduction to about 85% reduction, about 15% reduction to about 80% reduction, about 15% Reduced to about 75% reduced, about 15% reduced to about 70% reduced, about 15% reduced to about 65% reduced, about 15% reduced to about 60% reduced, about 15% reduced to about 55% reduced, about 15% about 50% reduction, about 15% reduction to about 45% reduction, about 15% reduction to about 40% reduction, about 15% reduction to about 35% reduction, about 15% reduction to about 30% reduction, about 15% about 25% reduction, about 15% reduction to about 20% reduction, about 20% reduction to about 99% reduction, about 20% reduction to about 95% reduction, about 20% reduction to about 90% reduction, about 20% about 85% reduction, about 20% reduction to about 80% reduction, about 20% reduction to about 75% reduction, about 20% reduction to about 70% reduction, about 20% reduction to about 65% reduction, about 20% Reduced to about 60% reduced, about 20% reduced to about 55% reduced, about 20% reduced to about 50% reduced, about 20% reduced to about 45% reduced, about 20% reduced to about 40% reduced, about 20% Reduced to about 35% reduced, about 20% reduced to about 30% reduced, about 20% reduced to about 25% reduced, about 25% reduced to about 99% reduced, about 25% reduced to about 95% reduced, about 25% about 90% reduction, about 25% reduction to about 85% reduction, about 25% reduction to about 80% reduction, about 25% reduction to about 75% reduction, about 25% reduction to about 70% reduction, about 25% Reduced to about 65% reduced, about 25% reduced to about 60% reduced, about 25% reduced to about 55% reduced, about 25% reduced to about 50% reduced, about 25% reduced to about 45% reduced, about 25% about 40% reduction, about 25% reduction to about 35% reduction, about 25% reduction to about 30% reduction, about 30% reduction to about 99% reduction, about 30% reduction to about 95% reduction, about 30% about 90% reduction, about 30% reduction to about 85% reduction, about 30% reduction to about 80% reduction, about 30% reduction to about 75% reduction, about 30% reduction to about 70% reduction, about 30% Reduced to about 65% reduced, about 30% reduced to about 60% reduced, about 30% reduced to about 55% reduced, about 30% reduced to about 50% reduced, about 30% reduced to about 45% reduced, about 30% Reduced to about 40% reduced, about 30% reduced to about 35% reduced, about 35% reduced to about 99% reduced, about 35% reduced to about 95% reduced, about 35% reduced to about 90% reduced, about 35% Reduced to about 85% reduced, about 35% reduced to about 80% reduced, about 35% reduced to about 75% reduced, about 35% reduced to about 70% reduced, about 35% reduced to about 65% reduced, about 35% Reduced to about 60% reduced, about 35% reduced to about 55% reduced, about 35% reduced to about 50% reduced, about 35% reduced to about 45% reduced, about 35% reduced to about 40% reduced, about 40% about 99% reduction, about 40% reduction to about 95% reduction, about 40% reduction to about 90% reduction, about 40% reduction to about 85% reduction, about 40% reduction to about 80% reduction, about 40% Reduced to about 75% reduced, about 40% reduced to about 70% reduced, about 40% reduced to about 65% reduced, about 40% reduced to about 60% reduced, about 40% reduced to about 55% reduced, about 40% Reduced to about 50% reduced, about 40% reduced to about 45% reduced, about 45% reduced to about 99% reduced, about 45% reduced to about 95% reduced, about 45% reduced to about 90% reduced, about 45% Reduced to about 85% reduced, about 45% reduced to about 80% reduced, about 45% reduced to about 75% reduced, about 45% reduced to about 70% reduced, about 45% reduced to about 65% reduced, about 45% about 60% reduction, about 45% reduction to about 55% reduction, about 45% reduction to about 50% reduction, about 50% reduction to about 99% reduction, about 50% reduction to about 95% reduction, about 50% about 90% reduction, about 50% reduction to about 85% reduction, about 50% reduction to about 80% reduction, about 50% reduction to about 75% reduction, about 50% reduction to about 70% reduction, about 50% about 50% to about 60% reduction about 50% to about 55% reduction about 55% to about 99% reduction about 55% to about 95% reduction about 55% about 55% to about 85% reduction, about 55% to about 80% reduction, about 55% to about 75% reduction, about 55% to about 70% reduction, about 55% Reduced to about 65% reduced, about 55% reduced to about 60% reduced, about 60% reduced to about 99% reduced, about 60% reduced to about 95% reduced, about 60% reduced to about 90% reduced, about 60% about 60% to about 80% reduction, about 60% to about 75% reduction, about 60% to about 70% reduction, about 60% to about 65% reduction, about 65% about 99% reduction, about 65% reduction to about 95% reduction, about 65% reduction to about 90% reduction, about 65% reduction to about 85% reduction, about 65% reduction to about 80% reduction, about 65% Reduced to about 75% reduced, about 65% reduced to about 70% reduced, about 70% reduced to about 99% reduced, about 70% reduced to about 95% reduced, about 70% reduced to about 90% reduced, about 70% reduced to about 85% reduced, reduced about 70% reduced to about 80% reduced, reduced about 70% reduced to about 75% reduced, reduced about 75% reduced to about 99% reduced, reduced about 75% reduced to about 95% reduced, about 75% Reduced to about 90% reduced, about 75% reduced to about 85% reduced, about 75% reduced to about 80% reduced, about 80% reduced to about 99% reduced, about 80% reduced to about 95% reduced, about 80% Reduced to about 90% reduced, about 80% reduced to about 85% reduced, about 85% reduced to about 99% reduced, about 85% reduced to about 95% reduced, about 85% reduced to about 90% reduced, about 90% reduction to about 99% reduction, about 90% reduction to about 95% reduction, or about 95% reduction to about 99% reduction) (e.g., compared to the volume of one or more solid tumors prior to treatment or at initiation of treatment) ). In some embodiments, these methods determine the risk of developing metastasis (e.g., in a subject prior to treatment or in a similar subject or subject population receiving a different treatment, or the risk of developing one or more additional metastases reducing the risk of developing metastasis or developing one or more additional metastases in a subject (e.g., from about a 1% reduction to about a 99% reduction, or any portion of this range as described herein) any of the ranges).

In some embodiments, these methods can result in treatment of metabolic disease in a subject. In some embodiments, the treatment of metabolic disease is, for example, one or more (eg, 2, 3, 4, 5, or 6) improved glucose tolerance, improved glucose utilization, diabetic bone Reduced severity or progression of arthritis, reduced severity or progression of skin lesions, reduced severity or progression of ketosis, reduced production of autoantibodies against pancreatic islet cells, increased insulin sensitivity, decreased mass, and obesity. can result in a decrease in the degree index. A subject's response to treatment can be monitored by determining fasting glucose or glucose tolerance according to standard techniques. Typically, according to this method, the blood glucose level is adjusted to achieve a blood glucose level characterized by a fasting blood glucose level of less than 100 mg/dL or a 2-hour oral glucose tolerance test value of 75 g of less than 140 mg/dL. is reduced to In some embodiments, the response to treatment includes blood pressure, body mass index, PPAR-γ function, lipid metabolism, glycated hemoglobin (H1c), and renal function, prediabetes, new-onset diabetes, or active Determining other factors associated with diabetes may be included.

In some embodiments, these methods include biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that manifest during the development of the disease. , can eliminate or reduce the risk of, reduce the severity of, or delay the development of neurodegenerative diseases.

In some embodiments, effective treatment of skin disorders improves skin condition, including skin color, moisture, temperature, texture, mobility and elasticity, and skin lesions, compared to skin condition prior to treatment. It can be assessed by any method described herein or known in the art, including examining.

In some embodiments, effective treatment of autoimmune diseases is described herein, comprising analysis of freshly isolated PBMCs for complete blood counts, total Ig levels, and serum autoantibody titers. or by any method known in the art.

In some embodiments, effective treatment of frailty disease includes bone mineral density, bone architecture and shape, biomedical markers of bone turnover, vitamin D measurements, Karnofsky performance status, and ECOG score; and by any method described herein or known in the art, including monitoring responsiveness to vaccination.

A method of killing senescent cells or reducing the number of senescent cells in a subject Killing senescent cells (e.g., any of the exemplary types of senescent cells described herein or known in the art) or A method of killing senescent cells 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 (e.g., Also provided herein are methods comprising administering an NK cell activator (any of the NK cell activators described herein or known in the art).

Killing senescent cells (e.g., any of the exemplary types of senescent cells described herein or known in the art) or killing senescent cells in a subject in need of reducing the number of senescent cells or reducing the number of senescent cells, wherein the subject is administered a therapeutically effective amount of one or more NK cells (e.g., any of the NK cells described herein or known in the art) Also provided herein is a method comprising administering ).

Some embodiments of these methods include obtaining resting NK cells and contacting the resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators, wherein Contacting results in the generation of activated NK cells that are then administered to the subject. In some examples of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some examples of these methods, the resting NK cells are haploidentical NK cells obtained from the subject. In some examples of these methods, the resting NK cells are allogeneic resting NK cells. In some examples of these methods, the resting NK cells are induced NK cells. In some examples of any of these methods, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

In some examples of these methods, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically defined liquid culture medium. Some examples of these methods include isolating activated NK cells (and administering a therapeutically effective amount of the activated NK cells to a subject, e.g., one of the subjects described herein). further administering to either). In some embodiments of these methods, the contacting step is carried out for a period of time from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of these methods, the senescent cells are senescent cancer cells, senescent monocytes, senescent lymphocytes, senescent astrocytes, senescent microglia, senescent neurons, senescent tissue fibroblasts, senescent skin fibroblasts , senescent keratinocytes, or other differentiated tissue-specific dividing functional cells. 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 an age-related disease or condition (e.g., any of the age-related diseases or conditions described herein or known in the art). have been identified or diagnosed as having In some embodiments of any of the age-related diseases or conditions described herein, the age-related disease or condition is cancer, autoimmune disease, metabolic disease, neurodegenerative disease, cardiovascular selected from the group of diseases, skin diseases, progeria and frailty diseases.

Non-limiting examples of cancer include solid tumors, hematologic tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms. , multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL) ), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, Colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, 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 disease include coronary artery disease, atherosclerosis, and pulmonary arterial hypertension.

Non-limiting examples of skin disorders include wound healing, alopecia, wrinkles, senile lentigines, skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

Non-limiting examples of progeria include progeria and Hutchinson-Guilford progeria syndrome.

Non-limiting examples of frailty diseases include frailty, response to vaccination, osteoporosis, and sarcopenia.

In some embodiments of any of the age-related diseases or conditions described herein, the age-related disease or condition is age-related macular degeneration, osteoarthritis, fat wasting, chronic obstructive pulmonary disease , idiopathic pulmonary fibrosis, renal graft failure, liver fibrosis, bone loss, sarcopenia, age-related pulmonary tissue elastic loss, osteoporosis, age-related renal dysfunction, and chemically-induced renal dysfunction selected from the group of

In some embodiments of any of the age-related diseases or conditions described herein, the age-related disease or condition is type 2 diabetes or atherosclerosis.

In some embodiments of these methods, administering results in a reduction (e.g., at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 25% reduction in the number of senescent cells in the target tissue in the subject). % reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60% reduction, at least 65% reduction, at least 70% reduction, at least 75 % reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, or at least 95% reduction, or about 10% reduction to about 99% reduction (or any subrange of this range described herein) either)) (eg, 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 is adipose tissue, pancreatic tissue, liver tissue, lung tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle. tissue, and one or more of secondary lymphoid organ tissue.

In some embodiments of these methods, administering results in an increase in levels of IFN-γ, cytotoxic granule granzymes, and/or perforin in the subject (e.g., at least a 5% increase, at least a 10% increase, at least a 15% increase, at least 20% increase, at least 25% increase, at least 30% increase, at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80% increase, at least 85% increase, at least 90% increase, at least 95% increase, or at least 99% increase, or about 10% increase to about 500% increase (or any of the subranges of this range described herein)) (compared to levels in pre-treatment subjects or similar control subjects not receiving 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) is determined by immunostaining a biopsy sample. can do. In some embodiments of these methods, the number of senescent cells in a target tissue (e.g., any of the target tissues described herein) is determined by an age-related disease or condition in a subject (e.g., can be indirectly observed through improvement in one or more symptoms of age-related diseases or conditions described in the literature).

Senescent Cells Senescent cells exhibit important unique characteristics including changes in morphology, chromatin organization, gene expression, and metabolism. Several biochemical and functional properties associated with cellular senescence, such as (i) increased expression of p16 ^INK4a and p21 ^CIP1 , inhibitors of cyclin-dependent kinases, (ii) senescence, a marker of lysosomal activity (iii) appearance of senescence-associated heterochromatin foci and downregulation of Lamin B1 levels, (iv) resistance to apoptosis caused by increased expression of anti-apoptotic BCL family proteins, and (v) CD26 ( DPP4), CD36 (scavenger receptor), forkhead box 4 (FOXO4), and secretory carrier membrane protein 4 (SCAMP4) are upregulated. Senescent cells also express inflammatory properties, the so-called senescence-associated secretory phenotype (SASP). Through SASP, senescent cells operate in a cell-autonomous manner to enhance senescence (autocrine effect) and communicate with and modify the microenvironment (paracrine effect), a wide range of inflammatory cytokines (IL-6, IL-8), growth factors (TGF-β), chemokines (CCL-2), and matrix metalloproteinases (MMP-3, MMP-9). SASP factors can contribute to tumor suppression by triggering senescence surveillance, the immune-mediated clearance of senescent cells. However, chronic inflammation is also a known factor in tumorigenesis, and cumulative evidence indicates that chronic SASPs may also drive cancer metastasis and age-related diseases.

The secretory profile of senescent cells is context dependent. For example, mitochondrial dysfunction-associated senescence (MiDAS) induced by various mitochondrial dysfunctions in human fibroblasts led to the emergence of SASPs deficient in IL-1-dependent inflammatory factors. A decrease in the NAD+/NADH ratio activated AMPK signaling that induced MiDAS through activation of p53. As a result, p53 inhibited NF-κB signaling, a key inducer of pro-inflammatory SASPs. In contrast, cellular senescence caused by persistent DNA damage in human cells induced inflammatory SASP, which depended on activation of the ataxia-telangiectasia mutated (ATM) kinase, It was independent of p53 activation. Specifically, IL-6 and IL-8 expression and secretion levels were increased. It has also been demonstrated that cellular senescence caused by ectopically expressed p16 ^INK4a and p21 ^CIP1 induces a senescent phenotype in human fibroblasts without inflammatory SASP, and that growth arrest itself did not stimulate SASP. is shown.

One of the most defining features of aging is steady growth arrest. This is accomplished by two key pathways, p16 ^INK4 a/Rb and p53/p21 ^CIP1 , both of which are central to tumor suppression. DNA damage is associated with (1) increased deposition of γH2Ax (a histone coding gene) and 53BP1 (involved in the DNA damage response) in chromatin, which leads to activation of kinase cascades that ultimately lead to p53 activation, and (2) ) activation of p16INK4a and ARF (both encoded by CDKN2A) and P15INK4b (encoded by CDKN2B) (p53 induces transcription of cyclin-dependent kinase inhibitor (p21 ^CIP1 ), both p16INK4a and p15INK4b , which shuts off genes for cell cycle progression (CDK4 and CDK6). This ultimately leads to hypophosphorylation of the retinoblastoma protein (Rb) and cell cycle arrest in the G1 phase.

Selectively killing senescent cells has been shown to significantly improve the health span of mice in the context of normal aging and improve outcome of age-related disease or cancer therapy ( Ovadya, J Clin Invest. 128(4):1247-1254, 2018). In nature, senescent cells are normally eliminated by innate immune cells. Induction of senescence not only blocks potential proliferation and transformation of damaged/altered cells, but also mainly natural killer (NK) cells (such as IL-15 and CCL2) and macrophages (such as CFS-1 and CCL2). It also supports tissue repair through the production of SASP factors that function as chemoattractants for ). These innate immune cells mediate immune surveillance mechanisms to eliminate stressed cells. Senescent cells normally upregulate the NK cell-activating receptors NKG2D and DNAM1 ligands, which belong to a family of stress-inducible ligands, which are key components of the first-line immune defense against infectious diseases and malignancies. . Upon receptor activation, NK cells can specifically induce senescent cell death through their cytolytic mechanism. The role of NK cells in immunosurveillance of senescent cells has been reported 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 dysfunction of the mevalonate pathway (Ciaglia, Int J Cancer 142(1): 176-190, 2018). Endometrial cells undergo acute cellular senescence and do not differentiate into decidual cells. Differentiated decidual cells secrete IL-15, which recruits uterine NK cells to target and eliminate undifferentiated senescent cells, helping to remodel and rejuvenate the endometrium (Brighton, Elife 6). : e31274, 2017). Using a similar mechanism, during liver fibrosis, p53-expressing senescent hepatic satellite cells polarize resident Kupfer macrophages and newly infiltrating macrophages exhibiting senolytic activity to the proinflammatory M1 phenotype. distorted towards F4/80+ macrophages have been shown to play an important role in the clearance of mouse uterine senescent cells to maintain postpartum uterine function.

Senescent cells primarily recruit NK cells by upregulating ligands for NKG2D (expressed on NK cells), chemokines, and other SASP factors. An in vivo model of liver fibrosis shows effective clearance of senescent cells by activated NK cells (Krizhanovsky, Cell 134(4):657-667, 2008). Studies have investigated liver fibrosis (Krizhanovsky, Cell 134(4):657-667, 2008), osteoarthritis (Xu, J Gerontol A Biol Sci Med Sci 72(6):780-785, 2017), and Parkinson's disease. Various models have been described for studying aging, including 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 that involves phenotypic changes, resistance to apoptosis, and activation of injury-sensing signaling pathways. Cellular senescence was first described in cultured human fibroblasts that lost the ability to proliferate, reaching permanent arrest after approximately 50 population doublings (termed the Hayflick limit) (Hayflick et al., Exp. Cell Res. 25:585-621, 1961). A phenomenon of “replicative senescence” was observed in cultures of non-immortalized human fibroblasts. This is caused by progressive shortening of telomeres with each cell division and represents a physiological response that prevents genomic instability and the consequent accumulation of DNA damage (He et al., Cell 169(6):1000- 1011, 2017).

Aging is viewed as a stress response that can be induced by a wide range of endogenous and exogenous insults, including oxidative and genotoxic stress, DNA damage, telomere attrition, or oncogenic activation, mitochondrial dysfunction, or chemotherapeutic agents. (McHugh et al., J. Cell Biol. 217(1):65-77, 2018). This accelerated aging response, independent of telomere shortening, is known as premature aging. Aging is associated with various age-related complications, such as diabetes, osteoporosis, cardiovascular disease, dementia, neurodegenerative disease, renal failure, and sarcopenia. It is also interesting that aging is the greatest risk factor for cancer (McHugh et al., J. Cell Biol. 217(1):65-77, 2018; Childs et al., Nat. Rev. .Drug Discov.16(10):718-735, 2017).

Senescent cells remain metabolically active and can influence tissue hemostasis, disease and aging through their secretory phenotype (He et al., Cell 169(6):1000-1011, 2017). ). Aging is considered a physiological process and 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 healing and contributes to wound resolution. Perhaps one of the most important roles of aging is its role in suppressing tumorigenesis (von Kobbe, Cell Mol. Life Sci. 2018). However, the accumulation of senescent cells also promotes aging and age-related diseases. The senescent phenotype may also provoke a chronic inflammatory response, thereby enhancing the chronic inflammatory state and promoting tumor growth. The link between senescence and aging was originally based on the observation that senescent cells accumulate in aging tissue. The last decade has greatly expanded our understanding of the detrimental consequences of aging in aging and age-related medical conditions. The use of transgenic models has made it possible to systematically detect senescent cells in many age-related disease states. The development of genetic and senolytic drug strategies to selectively eliminate senescent cells has demonstrated that senescent cells can indeed play a causal role in aging and related pathologies.

Senescent cells exhibit important unique characteristics including changes in morphology, chromatin organization, gene expression, and metabolism. Several biochemical and functional properties associated with cellular senescence, such as (i) increased expression of p16 ^INK4a and p21 ^CIP1 , inhibitors of cyclin-dependent kinases, (ii) senescence, a marker of lysosomal activity (iii) appearance of senescence-associated heterochromatin foci and downregulation of Lamin B1 levels, (iv) resistance to apoptosis caused by increased expression of anti-apoptotic BCL family proteins, and (v) CD26 ( DPP4) (Kim et al., Genes Dev. 31(15):1529-1534, 2017), CD36 (scavenger receptor) (Chong et al., EMBO Rep. 19(6), 2018), forkhead box 4 (FOXO4) (Bourgeois et al., FEBS Lett. 592(12):2083-2097, 2018) and secretory carrier membrane protein 4 (SCAMP4) (Kim et al., Genes Dev. 32(13-14):909 -914, 2018), (vi) accumulation of lipofuscin, and (vii) expression of embryonic chondrocyte 1 and decoy death receptor 2. Senescent cells also express inflammatory properties, so-called SASPs. Through SASPs, senescent cells operate in a cell-autonomous manner to enhance senescence (autocrine effect) and communicate with and modify the microenvironment (paracrine effect), a wide range of inflammatory cytokines (IL-1α, IL-1β, IL-6, IL-8, TNF-α), growth factors (TGF-β, 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) (Milanovic et al., Nature 553(7686):96-100, 2018) . IL-1α is considered one of the key regulators of SASPs. Release of IL-1α by senescent cells propagates senescence to normal cells. IFNs can also induce senescence by inducing DNA damage in target cells. IGFB can regulate the insulin-like growth factor (IGF) pathway, and IGF can function as a potent inducer of aging. Secreted TGF-β as one of the SASP factors can induce and maintain the senescent phenotype and age-related morbidity in an autocrine/paracrine manner. Integrin β3, which is regulated by the Polycom protein CBX7, was upregulated during senescence and promoted senescence by activating TGF-β signaling in an autocrine/paracrine manner, enhancing SASP in human fibroblasts. . Furthermore, TGF-β-mediated accumulation of senescent cells has been suggested in idiopathic pulmonary fibrosis. A recent report showed that TGF-β signaling induced a reduction in H4K20me3 abundance, which impairs DNA damage repair, upregulates miR-29a/c in fibroblasts, and targets Suv4-20h. Down-regulation has been shown to restore and accelerate aging. This pathway contributes to cardiac aging in vivo, and inhibition of TGF-β signaling restored H4K20me3 and improved cardiac function in aged mice.

Matrix metalloproteinases (MMPs) are key components of SASPs, including MMP-1 and MMP-3, which can function as regulatory elements of aging. They can cleave IL-8, IL-1, VEGF, and other chemokines of the CXCL/CCL family. In addition, senescent cells secrete serine proteases such as urokinase or tissue-type plasminogen activator.

SASPs are also composed of non-polymeric elements such as nitric oxide and reactive oxygen species that can affect the phenotype of neighboring cells.

The secretory profile of senescent cells is context dependent. For example, mitochondrial dysfunction-associated senescence (MiDAS) induced by various mitochondrial dysfunctions in human fibroblasts led to the emergence of SASPs deficient in IL-1-dependent inflammatory factors (Wiley et al., Cell Metab.23(2):303-314, 2016). A decrease in the NAD+/NADH ratio activated AMPK signaling that induced MiDAS through activation of p53. As a result, p53 inhibited NF-κB signaling, a key inducer of pro-inflammatory SASPs (Salminen et al., Cell Signal. 24(4):835-845, 2012). In contrast, cellular senescence caused by persistent DNA damage in human cells induced inflammatory SASP, which depended on activation of the ataxia-telangiectasia mutated (ATM) kinase, It was independent of p53 activation (Rodier et al., Nat. Cell Biol. 11(8):973-979, 2009). Specifically, IL-6 and IL-8 expression and secretion levels were increased. It has also been demonstrated that cellular senescence caused by ectopically expressed p16 ^INK4a and p21 ^CIP1 induces a senescent phenotype in human fibroblasts without inflammatory SASP, and that growth arrest itself did not stimulate SASP. (Coppe et al., J. Biol. Chem. 286(42):36396-36403, 2011). These indicate that the aging phenotype plays an important role in regulating the properties of SASPs and their physiological and pathological consequences.

Thus, multiple components of SASP have the ability to promote senescence in nearby non-senescent cells in a paracrine manner in order to increase the total number of senescent cells. Through SASPs, senescent cells can also influence the tissue microenvironment through paracrine mechanisms, influence neighboring proliferating cells, and influence the recruitment and activation of immune cells in senescent tissues and tumors.

SASP factors can contribute to tumor suppression by triggering senescence surveillance, the immune-mediated clearance of senescent cells. However, chronic inflammation is also a known factor in tumorigenesis, and cumulative evidence indicates that chronic SASPs may also drive cancer and age-related diseases. Recently, it has also been shown that senescent cells influence neighboring cells by direct transfer of intercellular proteins (Biran et al., Genes Dev. 29(8):791-802, 2015). Proteins transferred from senescent cells to recipient neighboring cells caused the activation of signaling pathways in these cells, resulting in changes in cell behavior. Recent studies have shown that chemotherapy-induced senescent cancer cells phagocytosed neighboring senescent or non-senescent cancer cells. Phagocytosis also occurred in the presence of the cell death inhibitor p53. Ingested cells are degraded by lysosomes. Senescent cells that ate neighboring cells survived longer than those that did not. This suggests that metabolic components obtained from lysosomal digestion of neighboring cells are used to promote survival of senescent cells. Phagocytosis was primarily due to phagocytosis rather than the mechanism of action of entosis. It was proposed that cell cannibalism may affect cancer progression by supporting the SASP response. However, this newly acquired ability of chemotherapy-induced senescent cancer cells can directly promote or facilitate cancer cell metastasis by removing specific cells from the tumor microenvironment. have a nature. If normal cells were also found to be removed by senescent cells in aged tissue, this could directly cause tissue deterioration.

In summary, all components of SASP contribute to the local inflammatory environment and may contribute to inflammatory phenomena.

Most of the SASP components are regulated by activated B-cell nuclear factor kappa light chain enhancer (NF-κB), CCAAT/enhancer binding protein beta (CEBP/β), and mTOR. The transcription factor GATA4, which acts upstream of NF-κB, is also required for establishment of senescence and induction of SASP. Another regulator of SASPs is the bromodomain and extraterminal domain (BET) family member bromodomain-containing protein 4 (BRD4), which positively regulates the aging secretome and promotes aging immune clearance. SASP is also regulated by the tissue-specific signal transducer and activator of transcription 3 (STAT3). In addition, mixed lineage leukemia 1 (MLL1) has also been reported to enable SASP, primarily by inducing genes required for DNA replication and DDR activation. Other SASP regulators include NOTCH1 and high mobility group B proteins (HMGB1 and HMGB2). Recent data also demonstrate that SASP can be regulated by the cGAS/STING pathway. cGAS is a DNA sensor that, via the adapter protein STING, triggers the transcription of genes that regulate cellular senescence and SASP.

One of the most defining features of aging is steady growth arrest. This is accomplished by the p53/p21 ^CIP1 p21 ^cip1 and p16 ^INK4a /Rb pathways (McHugh et al., J. Cell Biol. 217(1):65-77, 2018). DNA damage and/or DNA damage response (DDR) critically regulate these two pathways.

(1) p53/p21 ^CIP1 p21 ^cip1 : p53 plays a pivotal role in cellular senescence and its activation can be DDR dependent or DDR independent. In the case of telomere DDR dependence, overactivation of oncogenes and inactivation of tumor suppressors (oncogene-induced senescence, OIS) due to telomere attrition, DNA damage, and replication stress triggers DNA damage repair cascades. activated. DDR activates the stress sensors ataxia telangiectasia mutated kinase (ATM) or ataxia telangiectasia and Rad3-related (ATR) kinase. ATM/ATR then activates the p53/p21 ^CIP1 p21cip1 axis by phosphorylating both p53 and its ubiquitin ligase Mdm2, leading to stabilization of p53 levels. P53 is directly phosphorylated on Ser-15 and indirectly on Ser-20 via Chk1/2. A number of recent studies have also demonstrated that some OIS pathways can indeed bypass DDR and activate p53p35. These once again demonstrated an important role of p53 and p53-triggered senescence in suppressing tumorigenesis after initial mutational initiation.

Stabilization of the p53 protein upregulates p21 ^CIP1 . p21 ^CIP1 p21 ^cip1 , a member of the mammalian cyclin-dependent kinase (CDK) inhibitor family, p21 ^cip1 is required for cell cycle arrest by p53 at the G1/S or G2/M checkpoints. p21 ^CIP1 p21cip1, encoded by the CDKN1A gene located on human chromosome 6, is a potent cyclin-dependent kinase inhibitor (CKI). It binds to and inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes and functions as a regulator of cell cycle progression in G1 and S phases. p21 ^CIP1 p21 ^cip1 also mediates gene expression regulation of many p53 targets such as CDC25C, CDC25B and survival, mainly through E4F4 complex recruitment. p21 ^CIP1 p21 ^cip1 also promotes senescence through inhibition of apoptosis. It binds many apoptotic drugs, including many caspases. P21 ^CIP1 P21cip1 knockout in senescent cells causes programmed cell death through the caspase activation cascade. p21 ^CIP1 p21cip1 can also induce senescence independent of p53 activity. Chk2 was shown to induce p21cip1 expression in p53-deficient cell lines and contribute to Chk2-mediated senescence.

(2) p16 ^INK4a /Rb: Three tumor suppressors are present at the INK4/ARF locus: p16 ^INK4a and ARF are both encoded by the CNDN2A gene and p15 ^INK4b by the CDKN2B gene. p15 ^INK4b and p16 ^INK4a , like p21 ^CIP1 , are CDKIs that affect the cell cycle by binding to and inhibiting CDK4 and CDK6. In contrast, ARF inhibits MDM2, thereby allowing crosstalk with the p53/p21 ^CIP1 pathway. The INK4/ARF locus acts as an aging sensor. In young normal cells, deposition of repressive H3K27me3 marks epigenetically silences the INK4/ARF locus. Methylation of H3K27 is regulated by the Polycom repressive complexes, PRC2 and PRC3. Disruption of PRC1 or PRC2 activity by depleting the expression of some components represses p16 ^INK4a and induces senescence. During senescence, the H3K27 histone demethylase JMJD3 plays a role in removing repression marks around the INK4/ARF locus, facilitating its induction. Induction of INK4/ARF can be observed in tissues during natural aging. In particular, p16 ^INK4a is considered a biomarker of aging.

In summary, p53 induces transcription of the cyclin-dependent kinase inhibitor p21 ^CIP1 and together with both p16INK4a and p15INK4b shuts off genes for cell cycle progression (CDK4 and CDK6). This ultimately leads to hypophosphorylation of the retinoblastoma protein (Rb) and cell cycle arrest in the G1 phase (McHugh et al., J. Cell Biol. 217(1):65-77, 2018). .

The p53/p21 ^CIP1 pathway appears to play a key role in initiating senescence, whereas pathways involving the p16 ^INK4a and RB family appear to play a central role in maintaining senescence. This was suggested by the observation of decreased p53 levels after induction of senescence, whereas p16 ^INK4a levels remain steadily elevated. Downregulation of p53 in senescent cells has also been shown to have differential effects depending on p16 activity. p53 is successful in inducing replication and cell proliferation in cells with low levels of p16INK4a, but not in cells with high ^p16INK4a activity. These findings suggest that activation of the p16 ^INK4a /Rb pathway is involved in drawing the line between two distinct stages of aging. The early reversible step is governed by p53 activity and the irreversible step is induced by the p16 ^INK4a /Rb pathway.

Recently, the cGAS-cGAMP-STING pathway has emerged as an important link from DNA damage to inflammation, cellular senescence, and cancer (Tuo et al., J. Exp. Med. 215(5):1287-1299 , 2018). This pathway detects cytoplasmic DNA after DNA damage and activates type I IFNs and other cytokines. Both DNase2 and TREX1 rapidly remove cytoplasmic DNA fragments emanating from the nucleus of presenescent cells, but expression of these DNases is downregulated in senescent cells, resulting in cytoplasmic accumulation of nuclear DNA. This causes aberrant activation of the cGAS-STING cytoplasmic DNA sensor and triggers SASP through induction of IFN-β (Takahashi et al., Nature Comm. 9:1249, 2018).

Transforming growth factor-β (TGF-β) is an evolutionarily conserved superfamily of cytokines that mediate a diverse range of signaling functions to effect tissue-specific control of cell differentiation and proliferation. offer. It also promotes or protects cell death, promotes extracellular matrix protein expression, cell motility and invasion, and regulates cell metabolism.

The human TGF-β family includes 33 genes encoding homodimeric or heterodimeric secreted cytokines. Family members include activins, bone morphogenetic proteins, growth differentiation factors, Müllerian inhibitors, nodules and TGF-β. TGF-β family proteins are synthesized as precursor molecules consisting of a signal peptide, a prodomain (called latent associated peptide (LAP) for TGF-β), and a mature polypeptide. Removal of the short N-terminal signal peptide allows protein folding, glycosylation, and processing at subsequent biosynthetic steps during transport from the endoplasmic reticulum to the Golgi apparatus. Dimerization by disulfide bonds is followed by proteolytic cleavage of the polypeptide by a furin family protease, resulting in a long N-terminal dimer and disulfide-linked LAP and a short C-terminal dimer disulfide. Bound mature TGF-β is formed. LAP and mature TGF-β remain bound to each other and form a cryptic form of the ligand called the small latent complex (SLC), and structural analysis of this cryptic TGF-β form suggests that LAP It is shown to directly cover key amino acids of the C-terminal dimer that are later used to interact with signaling receptors and lead to inactivation of the mature TGF-β dimer. Concomitant with processing of the TGF-β polypeptide is cross-linking of the N-terminal LAP via disulfide bonds to another secreted protein, the latent TGF-β binding protein (LTBP), forming a large latent complex (LLC). ). LTBP is an extracellular protein that, when secreted, mediates LLC deposition into the extracellular matrix (EMC) through its ability to crosslink other proteins of the ECM such as fibronectin and fibrillin. LTBP thus provides a scaffolding unit that tethers potential TGF-β to the ECM. Three potential complexes of TGF-β require an activation mechanism to release the mature ligand. However, only activation of the TGF-β1 complex has been well characterized. Potential extracellular potential TGF-β activation in physiologically relevant settings, including proteolysis, low pH, reactive oxygen species, binding to other proteins, and mechanical deformation by shear or integrin-mediated cell pulling. The diverse modes of simplify tissue-selective mechanisms that may depend on signaling context. In various circumstances, one side of the LAP of SLC is covalently cross-linked via the Cys33 residue to the 8-Cys domain of LTBP, which is then linked to the ECM. This resistance allows for the conformational change of the latent TGF-β complex to pull the other end of LAP through the integrins, particularly αvβ1, αvβ6, and αvβ8, thus releasing active TGF-β from LLC. β1 release results. Various proteases also provide potential TGF-β activation. A number of studies strongly suggest that potential TGF-β1 physiological activation requires the combined activity of integrins and proteases. Latent TGF-β complexes have also been found to be disulfide-linked to a membrane-bound protein named GARP, also known as LRRC32, or the closely related LRRC33, instead of being associated with LTBP. GARP is expressed primarily on immune cells such as regulatory T cells (Treg). GARP's function has been extensively studied in Tregs, where it forms a complex with the αvβ8 integrin to release active TGF-β from the surface of the cell. GARP has been shown to be involved in enhancing Treg-mediated peripheral resistance. It has also been shown in platelets that thrombin cleaves GARP, releasing active TGF-β1 from the GARP-LAP-TGF-β1 complex.

Upon activation from LAP, all three TGF-β isoforms are mediated by the dimeric TGF-β type I receptor (RI) alk5 (also known as TβR1) and the dimeric TGF-β type II receptor (RII) TGFβRII. It acts through the same heteromeric transmembrane TGF-β receptor complex that is formed. TGF-β first binds to the homodimeric TGFβRII. This interaction causes a conformational adaptation between the ligand and TGFβRII such that a new high-affinity binding site for TGFβRI is formed at the ligand-TGFβRII interface. Upon recruitment of the two units of TGFβRI, the type II receptor kinase phosphorylates serine and threonine residues within the juxtamembrane subdomain of TGFβRI, which is characterized by a short glycine- and serine-rich motif (GS), It induces a conformational change that releases the immunophilin FKBP12 from the GS domain. This dissociation relieves the inhibitory interaction of the kinase and GS domains and activates the type I receptor kinase. Upon ligand-induced receptor activation, TGFβRI activates the effector SMAD through phosphorylation of two C-terminal serine residues. Specifically, type I receptors are two different SMAD proteins, SMAD2 and SMAD3, in the case of TGF-β (and other family members such as activin and nodal), or BMPs (some GDFs and other ligands). members) phosphorylates three different SMAD proteins, SMAD1, SMAD5, and SMAD8. These "receptor-activated SMADs" (R-SMADs) then dissociate from their receptors and bind SMAD4 to form complexes that translocate into the nucleus, producing high-affinity DNA-binding transcription factors and co-regulators. to activate or repress target genes. The SMAD complex not only directs gene transcription leading to secondary gene expression changes, but also controls mRNA splicing, miRNA expression and processing, and epigenetic changes. Due to the additional diversity of SMAD complex formation, the SMAD signaling pathway is a highly versatile, context-dependent and subtle pathway that regulates gene expression.

In addition to canonical SMAD signaling, TGF-β can also modulate downstream cellular responses through other signal transducers in a context-dependent manner. These include the ERK MAP kinase pathway, JNK and p38 MAP kinase pathway (via TAK1), PI3-AKT pathway, JAK-STAT pathway, Rho-(like) GTPase pathway, TGF-β type I receptor domain signaling pathway . It is now well known that the wide functional diversity and dependence of SMADS lies on these non-canonical pathways.

Because TGF-β controls the differentiation of most, if not all, cell lineages and regulates many aspects of cell and tissue homeostasis, deregulation of TGF-β signaling is associated with developmental abnormalities and diseases. leads to Accumulating evidence suggests that impaired TGF-β signaling and regulation of TGF-β ligands in specific cell types contributes to cellular senescence, cell degeneration, tissue fibrosis, inflammation, decreased regenerative capacity, and metabolic dysfunction. indicate that

Secreted as one of the SASP factors, TGF-β1 induces or accelerates and maintains the senescent phenotype in various cell types, including fibroblasts, bronchial epithelial cells, and cancer in an autocrine/paracrine manner. be able to. Integrin β3, which is regulated by the Polycom protein CBX7, was upregulated during senescence and promoted senescence by activating TGF-β signaling in an autocrine/paracrine manner, enhancing SASP in human fibroblasts. . Furthermore, TGF-β1-mediated accumulation of senescent cells has been suggested in idiopathic pulmonary fibrosis. A recent report showed that TGF-β1 signaling induced a reduction in H4K20me3 abundance, which impairs DNA damage repair, upregulates miR-29a/c in fibroblasts, and targets Suv4-20h. Down-regulation has been shown to restore and accelerate aging. This pathway contributes to cardiac aging in vivo, and inhibition of TGF-β signaling restored H4K20me3 and improved cardiac function in aged mice.

TGF-β1 functions as a driver of senescence, activation of the NF-κB signaling pathway by reactive oxygen species (ROS) stimulation, and SASP factors including plasminogen activator inhibitor type 1 (PAI-1, serpin 1). induces senescence of vascular smooth muscle cells (VSMCs) through the expression of PAI-1 is not only a biomarker of cellular senescence, but it is necessary and sufficient for replicative senescence downstream of p53 and is a key inducer of the senescence program. Evidence exists to suggest the existence of a PAI-1/TGF-β1 positive feed-forward mechanism, providing a model in which elevated tissue levels of TGF-β1 during the emergence of the senescent phenotype stimulate PAI-1 expression. . Second, it enhances continued TGF-β1 synthesis, which promotes maintenance and possibly expansion of aged VSMC populations (Seo et al., Am. J. Nephrol. 30:481-490, 2009).

Aging is well known to have tumor suppressive effects that slow clinical progression after chemotherapy. Over the past decade, great advances have been made in understanding the biology of aging, particularly those with tumor suppressor properties that may lead to pro-oncogenic effects on adjacent cancer cells, stroma and vasculature in the tumor microenvironment. transformed into a highly complex, dynamic and interactive one (Hoare et al., Ann. Rev. Cancer Biol. 2:175-194, 2018). A highly sophisticated study by Milanovic showed that senescent cells also upregulated key stem cell-associated transcripts in primary B-cell chronic leukemia patient samples (Milanovic et al., Nature 553 ( 7686):96-100, 2018). In patients with acute myeloid leukemia (AML), senescent cells have been shown to induce senescent phenotypes in stromal cells, and these stromal cells feed back to AML blasts via SASP. (Abdul-Aziz et al., Blood 133(5):446-456, 2019). Tumors seize a pathophysiologic program of growth regulation aimed at engaging in organ development or tissue repair, and instead of creating new mechanisms for tumor progression, they subvert this process for oncogenic performance. (Milanovic et al., Trends Cell Biol. 28(12):1049-1061, 2018). Epigenetic mechanisms have been described as responsible for the induction of senescence (H3K9 demethylase) and subsequent acquisition of stemness (H3K9 demethylase inhibition) (Yu et al., Cancer Cell 33(2):322-336). , 2018).

TGF-β1 induces epithelial-mesenchymal transition (EMT) by inducing the expression of specific transcription factors Snail and Zeb1/2. EMT provides migratory and invasive behavior to cells by altering cell adhesion. This process involves loss of epithelial and acquired function leading to motility and invasiveness. EMT is an important process leading to cancer cell progression and metastasis.

As an immunosuppressive cytokine, TGF-β1 inhibits the function and development of innate and adaptive immune systems such as macrophages, natural killer cells, dendritic cells and T cells. Recent in vivo studies have demonstrated that exposure to tissue- or tumor-derived TGF-β1 promotes the conversion of circulating NK cells to an innate lymphocyte-I (ILC-I)-like phenotype, leading to cytotoxicity It is characterized by reduced potency and the acquisition of several ILC1-associated surface markers. Interestingly, TGF-β1 also synergizes with IL-15 through the MAPK pathway to promote conversion of human NK cells to an ILC-1-like phenotype. TGF-β also suppresses human NK cell metabolism and suppresses NK cell cytotoxicity through canonical signaling pathways. TGF-β1 also stimulates regulatory T cells that suppress the function of other lymphocytes. These suppressive functions give TGF-β1 one of the many hallmarks of cancer that evades immune destruction.

Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas, with a 5-year survival rate of 7% and a median survival time of less than 11 months, with an extremely poor prognosis. PDAC is highly refractory to all available anti-tumor pharmacological options. This is the result of a strong fibrogenic response associated with PDAC progression, indicating strong activation of pancreatic stellate cells and the formation of a dense extracellular matrix, resulting in poor tumor perfusion. , an impenetrable barrier to intravenously infused anticancer or chemotherapeutic agents is formed. TGF-β1 contributes to PDAC fibrogenesis by promoting the conversion of fibroblasts or endothelial cells to myofibroblasts, also known as cancer-associated fibroblasts (CAFs). Aggression is further amplified by immune cells and fibroblasts infiltrating the tumor microenvironment that produce high levels of TGF-β. TGF-β induces pro-angiogenic factors such as vascular endothelial growth factor, allowing PDAC progression, invasion and metastasis. Acyl-CoA synthase long chain 3 (ACSL3) was found to be upregulated in PDAC and correlates with increased fibrosis. Reduction of PAI-1 secretion from tumor cells by Acsl3 knockout significantly reduces tumor fibrosis and tumor-infiltrating immunosuppressive cells and increases cytotoxic T-cell infiltration in mice. This study also found that PAI-1 expression in PDAC was positively correlated with markers of fibrosis and immunosuppression and predicted poor patient survival. This ACSL3-PAI-1 is an important component, a mediator of cellular senescence, and is regulated by TGF-β1, which mediates the tumor-stromal crosstalk that promotes pancreatic cancer progression. It is thought to play a role in the signaling axis.

In fibrotic diseases, excessive deposition of extracellular matrix (EMC) proteins compromises tissue integrity and interferes with normal organ function. Fibrosis can occur in chronically injured tissues, but is most frequently observed in the kidney, liver, lung, and heart. Fibrosis is primarily caused by inflammatory cytokines, including interleukins and members of the TGF-β superfamily. Many of these ligands are expressed by infiltrating inflammatory cells that are attracted to damaged tissue. Overexpression of TGF-β1 induces fibrosis through activation of both canonical (SMAD-based) and non-canonical (non-SMAD-based) signaling pathways, resulting in activation of myofibroblasts, ECM overproduction of and inhibition of ECM degradation occurs. Activation of SMAD2/3 is associated with several fibrotic pathways, including collagen (COL1A1, COL3A1, COL5A2, COL6A1, COL6A3, and COL7A1), PAI-1, various proteoglycans, integrins, connective tissue growth factors, and matrix metalloproteases. Regulates the expression of promoting genes. This results in excessive deposition of ECM and compromises local tissue architecture.

Signaling through the SMAD pathway is thought to play a central role in TGF-β fibrogenesis, but reactive oxygen species (ROS) also regulate TGF-β through various pathways, including the SMAD pathway. There is emerging evidence implicating it in regulating signal transduction. TGF-β1 increases mitochondrial ROS production in various cell types that mediate TGF-β-induced cell apoptosis, senescence, EMT, fibrotic gene expression, and myofibroblast differentiation. TGF-β is a group of heme-containing transmembrane proteins important for ROS production in both phagocytic and non-phagocytic cells, several NADPH oxidase (Nox) enzymes (Nox1, Nox2, and Nox4 in different types of cells). including ). Nox4-derived ROS mediate the fibrogenic effects of TGF-β, including fibroblast activation/myofibroblast differentiation, epithelial and endothelial cell apoptosis, EMT, and fibroblast/fibroblast-promoting gene expression. Mediate. Increased Nox4 expression has also been detected in fibrotic diseases, including IPF, which correlates with increased expression of the myofibroblast marker α-SMA, further supporting a role for Nox4 in fibrotic diseases. Several pathways have been shown to be involved in the induction of Nox4 by TGF-β. These include the SMAD, PI3K, MAPK, and RHOA/ROCK pathways.

Emerging evidence suggests that there is crosstalk between mitochondria and NADPH oxidase. Mitochondrially-derived ROS contribute to increased NOX expression in response to TGF-β, whereas NOX-generated ROS cause mitochondrial dysfunction and increase mitochondrial ROS production. Cross-talk between mitochondria and Nox enzymes has also been shown to mediate the pro-fibrogenic effects of TGF-β. A feed-forward interaction between mitochondria and Nox4 in TGF-β-induced ROS production has been implicated (Jain et al., Journal of Biological Chemistry. 288:770-777, 2013).

By 2030, more than 20% of the population will be over 65 (see census.gov/content/dam/Census/library/publications/2014/demo/p23-212.pdf) and about 40% will be obese (Finkelstein et al., Am. J. Prev. Med. 42(6):563-570, 2012). Metabolic disorders affect the ability of cells to carry out important processes including the transport or processing of proteins, carbohydrates and lipids. Aging and obesity are major risk factors for chronic diseases that predispose to diseases such as diabetes, cardiovascular disease, and fatty liver, all of which are leading causes of death and thus raise significant public health concerns. (Must et al., "The Disease Burden Associated with Overweight and Obesity," In: Feingold KR, Anawalt B., Boyce A., et al., eds., Endotext, South Dartmouth (MA), 2000; Martin et al. ., Nat. Rev. Cardiol. 14(3):132, 2017).

Excessive caloric intake promoted oxidative stress in adipose tissue in mice, resulting in hallmarks of type 2 diabetes, concomitant with the expression of senescence markers such as p53 and beta-galactosidase in mice (Minamino et al., Nat. Med. .15(9):1082-1087, 2009). Aging also accelerated the biological loss of adipose tissue by impeding adipogenic differentiation (Mitterberger et al., Gerontol. A Biol. Sci. 69(1):13-24, 2014). Another recent study showed that obesity-induced aging can cause anxiety and neurogenesis disorders by increasing fat deposits in the brain, and that clearance of these senescent cells is reduced by obesity in mice. It has been shown to lead to improvement in induced anxiety-like behavior (Ogrodnik et al., Cell Metab. 29(5):1061-1077, 2019). Other studies have shown that obesity also impairs immune cell function. Effector function of NK cells 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 impairment in obesity is independent of age, with similar defects observed in young and old obese individuals (Tobin et al., JCI Insight 2 (24) : e94939, 2017; Michelet et al., Nat. Immunol. 19(12): 1330-1340, 2018).

In mice, increased caloric intake leads to lipid deposition in blood vessels, and monocytes are recruited to phagocytose these lipids, ultimately into frothy macrophages that accumulate in the subendothelial space and cause atherosclerotic plaques. (Bennett et al., Nat. Rev. Cardiol. 14(3):132, 2017; Katsuumi et al., Front. Cardiovasc. Med. 5:18, 2018). Mice fed a Western high-fat diet (42% fat-derived calories) also showed that senescent cell burden was directly proportional to plaque (lipid-laden macrophage) formation. Successful elimination of these senescent cells in transgenic mice greatly reduced plaque formation (Childs et al., Science 354(6311):472-477, 2016).

Other factors associated with age, obesity, and changes in glucose levels, growth hormone (IGF), can lead to diabetes (Palmer et al., Diabetes 64(7):2289-2298, 2015). Upregulation of senescence markers such as p53 in mice fed a high-fat diet correlated with insulin resistance, whereas inhibition of p53 activity in adipose tissue led to decreased senescence markers and insulin resistance in a mouse model. (Minamino et al., Nat. Med. 15(9):1082-1087, 2009). Concurrently, senescence of pancreatic β-cells has been shown to contribute to type 2 diabetes in obese mice (Sone et al., Diabetologia 48(1):58-67, 2005).

Hypothalamic production of TGF-β is excessive under high-fat diet conditions. This causes hypothalamic inflammation, hyperglycemia, and glucose intolerance. This data suggests that excessive amounts of TGF-β induce a hypothalamic RNA stress response and promote IκBα mRNA decay. 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 system.

Aging is a major risk factor for developing many neurodegenerative diseases. Accumulation of senescent cells in the nervous system is shown in aging and neurodegenerative diseases and may predispose to the development of or exacerbate the course of neurodegenerative conditions (Kritsilis et al., Int. J. Mol. Sci 19 (10:2937, 2018) Cellular senescence can interfere with cellular function by: 1. Promoting chronic inflammation (Huell et al., Acta Neuropathol. 89(6):544-551). Nelson et al., Aging Cell 11(2):345-349, 2012), 2. Exhaustion of neuronal regeneration (Cipriani et al., Cereb. Cortex 28(7):2458-2478, 2018), 3 4. Blood-brain barrier dysfunction (Yamazaki et al., Stroke 47(4): 1068-1077, 2016) Studies have shown the accumulation of Aβ peptides, including amyloid plaques and misfolded tau proteins, in Alzheimer's disease (AD), the most common neurodegenerative disease in humans ( Musi et al., Aging Cell 17(6): e12840, 2018).These changes ultimately affect neurons, causing cognitive impairment and neurodegeneration.Astrocytes cultured from AD patients are well known. showed high expression of CDKi p16INK4A, MMP-1 and IL-6, which are known senescence markers (Bhat et al., PLoS One7(9): e45069, 2012; Myung et al., Age 30(4): 209-215, 2008).A clinical trial targeting amyloid protein was disappointing (Mehta et al., Expert Opin. Invest. Drugs 26(6):735-739, 2017). The presence of senescent cells has been shown to be responsible for neuropathy in animal models (Crews et al., Hum. Mol. Genet. 19(R1):R12-R20, 2010; Chinta et al., Cell Rep. .22(4):930-940, 2018). Studies in animal models reflective of human AD show encouraging results. Clearance of senescent cells in transgenic mice prevented abnormal accumulation of neurofibrillary tangles and tau protein in neurons and preserved cognitive function (Bussian et al., Nature 562(7728):578-582, 2018). ). Patients with Parkinson's disease (PD), the second most common neurodegenerative disease, exhibit loss of motor control due to loss of dopaminergic neurons in the substantia nigra. Astrocytes are the most abundant cell type within the CNS and are important for providing structural and metabolic support to neurons and also play a role in regulating the blood-brain barrier and blood flow. A recent landmark study demonstrated a senescent phenotype in astrocytes from postmortem brain samples from PD patients (Chinta et al., Cell Rep. 22(4):930-940, 2018). In this study, an animal model of PD induced by an environmental neurotoxin (palquat, which induces senescence via oxidative stress) was also developed, demonstrating neuropathology associated with PD. The authors showed that depleting senescent cells in transgenic mice abolished paraquat-induced neuropathology.

Aging of human skin consists of:1. endogenous (chronological), which is the result of physiological and genetic changes over time;2. Either exogenous, caused by exposure to external factors such as ultraviolet (UV) radiation, environmental toxins, and other agents that can induce DNA damage (Cavinato et al., Exp. Gerontol. .94:78-82, 2017). Among the changes that affect skin tissue with age, the loss of elastic properties caused by changes in elastin production, degradation and/or increased processing has a significant impact on tissue aesthetics and health (Wang et al., Front. Genet. 9:247, 2018). Acute UV exposure leads to sunburn, abnormal skin pigmentation, visible appearance of blood vessels under the skin (telangiectasia) and immunosuppression, whereas long-term exposure is associated with premature skin aging and malignant tumors. It may even lead to an onset risk (Rittie et al., Cold Spring Harb. Perspective 5(1):a015370, 2015). There is a direct correlation between the evolution of medicine and population growth, which is characterized by an increasing number of middle-aged and elderly people, and thus a great demand for anti-aging treatments (Weihermann et al., Int. J. Cosmet.Sci.39(3):241-247, 2017). UVB from sunlight is mutagenic and directly induces DNA damage during DNA replication. Photodamaged skin is characterized by the accumulation of amorphous elastic fibers in addition to disorganized dermal collagen. Studies have shown that this may be due to either impaired elasticity and fibrillin production or increased degradation of matrix metalloproteinases (MMPs) secreted by DNA-damaged senescent cells. (Pittayapruek et al., Int. J. Mol. Sci. 17(6):868, 2016). Production of reactive oxygen species (ROS) after UVB irradiation leads to activation of factors central to aging such as nuclear factor kappa (NF-κB) and mitogen-activated protein kinase (MAPK) (Pittayapruek et al., Int. J. Mol. Sci. 17(6):868, 2016). UVB irradiation can alter the TGF-β signaling pathway in human skin fibroblasts, mainly by decreasing the synthesis of transforming growth factor-β receptor II (TβRII) (Purohit et al., J. .Dermatol.83(1):80-83, 2016). Several studies have shown the presence of senescent cells in aged and UV-exposed skin both in vitro and in vivo. Keratinocytes and dermal fibroblasts have been extensively studied as models of photoaging expressing markers of aging such as p16 ^INK4asd , β-galactosidase, lamin B1 and senescence-associated secretory phenotype (SASP) factors (Waaijer et al., Aging 10(2):278-289, 2018; Dimri et al., Proc.Natl.Acad.Sci USA 92(20):9363-9367,1995; Ghosh et al., J. Invest.Dermatol.136(11):2133-2139, 2016). Since senescent cells are known to express NK ligands, inducing NK cells along with activation of other immune cells (regulatory T cells) eliminates senescent cells and maintains healthy skin. (Carr et al., Clin. Immunol. 105(2):126-140, 2002; Ali et al., Immunology 152(3):372-381, 2017).

Selective killing of senescent cells has been shown to significantly improve the healthy lifespan of mice in the context of normal aging, thus improving the outcome of age-related diseases or cancer therapy. There is growing interest in identifying compounds that can remove cells. In nature, senescent cells are normally eliminated by innate immune cells. Induction of senescence not only blocks potential proliferation and transformation of damaged/altered cells, but also mainly natural killer (NK) cells (such as IL-15 and CCL2) and macrophages (such as CFS-1 and CCL2). Tissue repair is also supported by the production of SASP factors (Munoz-Espin et al., Nat. Rev. Mol. Cell Biol. 15(7):482-496, 2014) that function as chemoattractants for . These innate immune cells mediate immune surveillance mechanisms to eliminate stressed cells. Senescent cells normally upregulate the NK cell-activating receptor NKG2D and DNAM1 ligand, which belong to the family of stress-inducible ligands, which are key components of the first line of immune defense against infectious diseases and malignancies. be. Upon receptor activation, NK cells can specifically induce senescent cell death through their cytolytic mechanism. The role of NK cells in immunosurveillance of senescent cells has been reported 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 gliomas stressed by dysfunction of the mevalonate pathway. cells (Ciaglia et al., Int. J. Cancer 142(1):176-190, 2018). In cancer, combination chemotherapy has been shown to upregulate senescence markers and NK ligands in KRAS-mutant lung tumors, suggesting that NK cells are required to target these cells. (Ruscetti et al., Science 362(6421):1416-1422, 2018). Endometrial cells undergo acute cellular senescence and do not differentiate into decidual cells. Differentiated decidual cells secrete IL-15, which recruits uterine NK cells to target and eliminate undifferentiated senescent cells, helping to remodel and rejuvenate the endometrium (Brighton et al. , Elife 6, 2017). Using a similar mechanism, during liver fibrosis, p53-expressing senescent hepatic satellite cells polarize resident Kupfer macrophages and newly infiltrating macrophages exhibiting senolytic activity to the proinflammatory M1 phenotype. distorted towards F4/80+ macrophages have been shown to play an important role in the clearance of mouse uterine senescent cells to maintain postpartum uterine function (Lujambio et al., Cell 153(2):449-460, 2013).

Strategies for senescent cell clearance fall into three main categories: senolytic agents, immunotherapy, and SASP inhibition (He et al., Cell 169(6):1000-1011, 2017). There is increasing body evidence suggesting the efficacy of senolytic agents to clear out senescent cells. Senolytic agents generally act by targeting the senescent cell anti-apoptotic pathway (SCAP), such as the BCL-2 protein family, the p53/p21 ^CIP1 p21 axis, PI3K/AKT, receptor tyrosine kinases, and HSP90 proteins. do. In mice, senolytic agents alleviate various conditions associated with the effects of senescent cells. So far, these have included effects on cardiac, vascular, metabolic, neurological, radiation-induced, chemotherapy-induced, renal, and pulmonary function, as well as motility and frailty in several animal models (Kirkland et al. al., EBioMedicine 21:21-28, 2017). Currently, more senolytic drugs are being developed. Recently, FOXO4-related peptides that inhibit the PI3K/AKT/p53/p21 pathway were reported and showed promising results in both in vitro human fibroblast and mouse models. Other senolytic agents include ABT-737 and ABT-263 (Tse et al., Cancer Res. 68(9):3421-3428, 2008), which act on the BCL-2 protein, and the BCL-XL pathway. Dasatinib and quercitin, which target tyrosine kinases, including targets A1331852 and A1155463 (Zhu et al., Aging (Albany NY) 9(3):955-963, 2017), show clearance of senescent cells ( Farr et al., Nat. Med. 23(9):1072-1079, 2017). BCL-2 family inhibitors can cause side effects such as neutropenia and thrombocytopenia. Since many senolytics are only in preclinical stages, potential side effects need to be studied before moving to clinical trials.

Blocking SASP factors is an alternative strategy to prevent the detrimental role of senescent cells. These factors include inflammatory chemokines and cytokines, growth factors, and matrix remodeling proteases. The central pathways involved in these effects are the NF-κB and C/EBPβ pathways. mTOR inhibitors such as rapamycin and its analogues can abrogate SASP by reducing the expression of membrane-bound IL-1α. Two other notable drugs used to inhibit the NF-κB and C/EBPβ pathways in vivo mouse models are metformin and ruxolitinib, respectively (Moiseeva et al., Aging Cell 12(3): 489-498, 2013; Xu et al., Proc. Natl. Acad. Sci. USA 112(46):E6301-6310, 2015). Other drugs such as siltuximab or tocilizumab block cytokines such as IL-6, another SASP factor. Again, as with the use of some senolytic agents, treatment with anti-inflammatory drugs can cause potential side effects (Karkera et al., Prostate 71(13):1455-1465, 2011). ). A recent phase I clinical trial using senolytic agents (dasatinib and quercetin) in patients with pulmonary fibrosis yielded inconclusive results (Justice et al., EBioMedicine 40:554-563). , 2019).

A third strategy that may be superior to the above strategies is immune-mediated intervention. As noted above, cells recruited to clear out senescent cells include NK cells, macrophages, and neutrophils. Senescent cells primarily recruit NK cells by upregulating ligands for NKG2D (expressed on NK cells), chemokines, and other SASP factors. An in vivo model of liver fibrosis shows effective clearance of senescent cells by activated NK cells (Krizhanovsky et al., Cell 134(4):657-667, 2008). Senescent cells resist NK cell-mediated clearance by upregulating the apoptosis-inhibiting decoy receptor DCR2 and limiting their clearance primarily by granzyme- and perforin-mediated pathways (Sagiv et al., Oncogene 32 (15): 1971-1977, 2013). Recent data indicate that NK cell lipid accumulation in obese individuals leads to a reduction in both their frequency and effector cytotoxic function, which is independent of age (Michelet et al., Nat. Immunol.19(12):1330-1340, 2018; Tobin et al., JCI Insight 2(24):e94939, 2017). NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) has been demonstrated in in vitro human senescent cells against dipeptidyl peptidase 4 (DPP4/CD26), a recently reported marker of senescence (Kim et al., Genes Dev. 31(15):1529-1534, 2017). Other strategies include using CAR-T cells to redirect the immune response against senescent cells (Grupp et al., N. Engl. J. Med. 368(16):1509-1518, 2013 Yousefzadeh et al., Nature, published online on May 12, 2021).

Studies have shown liver fibrosis (Krizhanovsky et al., Cell 134(4):657-667, 2008), osteoarthritis (Xu et al., J. Gerontol. A Biol. Sci. Med. Sci. 72 ( 6): 780-785, 2017), Parkinson's disease (Chinta et al., Cell Rep. 22(4): 930-940, 2018), obesity-induced anxiety (Ogrodnik et al., Cell Metab. 29(5) : 1061-1077, 2019), atherosclerosis (Childs et al., Science 354(6311): 472-477, 2016), and diabetes (Sone et al., Diabetologia 48(1): 58-67, 2005) have been described to study aging. One recent study showed that transplantation of in vitro senescence-induced cells into young mice resulted in physical dysfunction (Xu et al., Nat. Med. 24(8):1246-1256, 2018). . A remaining question is what types of therapies are effective in eliminating senescent cells in various tissues. Most of the 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). Baker et al., Nature 479(7372):232-236, 2011; Farr et al., Nat. Med.23(9):1072-1079, 2017; J. Gerontol.A Biol.Sci.Med.Sci.72(6):780-785, 2017;Bourgeois 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 have investigated this strategy in aging models (Krizhanovsky et al., Cell 134(4):657-667, 2008). Various clinical trials have shown that adoptive transfer of NK cells has been used successfully to treat various forms of cancer (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; : 134-148, 2018). Importantly, a recent clinical trial utilizing autologous ex vivo expanded NK cells in colon cancer patients (Li et al., Cytotherapy 20(1):134-148, 2018). The authors have shown that NK cell therapy combined with chemotherapy prevents relapse and prolongs survival with tolerable side effects (Li et al., Cytotherapy 20(1):134-148, 2018). Transfer of cytokine-activated NK cells with cytokines such as IL-15, IL-12, IL-18, IL-21 can be used as a potential immunotherapeutic strategy to eliminate senescent cells with minimal side effects (Romee et al. al., Blood 120(24):4751-4760, 2012; Song et al., Eur. J. Immunol.48(4):670-682, 2018). Furthermore, the safety of the use of NK cells in acute myeloid leukemia has been shown (Romee et al., Blood 120(24):4751-4760, 2012; Fehniger et al., Biol. Blood Marrow Transplant. 2018). . Another approach is to block circulating SASP factors such as TGF-β, IL-8, IL-6 (Ganesh et al., Immunity 48(4):626-628, 2018; Georgilis et al., Cancer Cell 34(1):85-102, 2018). The model of aging described above is ideal for testing these approaches. Therefore, more consideration needs to be given to strategies that avoid the unwanted side effects of using foreign compounds and drugs as a remedy for age-related medical conditions.

Cellular senescence is a series of progressive and phenotypically diverse cellular states acquired after initial growth arrest (Van Deursen, Nature 509(7501):439-446, 2014). Senescent cells are therefore a heterogeneous population of cells that share few core properties (Dou et al., Nature 550(7676):402-406, 2017). Therefore, it is difficult to identify targets for common senolytic drugs. This further hinders the goal of developing senolytic agents that selectively, safely and effectively eliminate senescent cells by systemic administration. As mentioned above, immune cells are effector cells for the natural elimination of senescent cells after they have fulfilled their physiological role (Brighton et al., Elife 6, 2017). Weakening of the immune system during the aging process allows senescent 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-β, a component of the SASP of senescent cells, plays a severe role in cellular senescence and senescence-related pathologies when overproduced in tissues (Tominaga et al., Int. J. .Mol.Sci.20(20), 2019). Methods for promoting and activating immune cells using complexes of common gamma chain cytokines and their cognate receptors, and amounts of active forms of TGF-β in aging tissue and tumor microenvironment using TGF-βRII Provided herein is a method of subcutaneously reducing , restoring the ability to reduce senescent cells, and effectively, selectively and safely reduce chronic inflammation in vivo.

In some embodiments of any of the methods described herein, the method rejuvenates aged immune cells in the subject (e.g., increases metabolic activity (e.g., increases oxidative phosphorylation, glycolysis, and increased oxygen consumption)), decreased levels of one or more of p16, p21, and SASP factors in aged immune cells in the subject, and decreased levels of aged immune cells in the subject. resulting in an increase in cytolytic activity, eg, compared to the subject's level prior to treatment. As used herein, the term "senescent immune cells", e.g., compared to control immune cells obtained from healthy subjects (non-immunocompromised subjects) who are less than half the age of the subject population's life expectancy, decreased metabolic activity (e.g. decreased oxidative phosphorylation, decreased glycolysis and decreased oxygen consumption), increased levels of one or more of the p16, p21 and SASP factors, and decreased cytolytic activity means an immune cell that has one or more of: Non-limiting examples of senescent immune cells include senescent NK cells, senescent NKT cells, senescent T cells, senescent B cells, senescent monocytes, senescent macrophages, senescent neutrophils, senescent Basophils, senescent eosinophils, Kupffer cells, and senescent microgial cells.

In some embodiments of any of the methods described herein, the method results in an increase in the naive to memory T cell ratio in the subject. In some embodiments of any of the methods described herein, the method results in a decrease in the ratio of CD4 ⁺ T cells to CD8 ⁺ T cells in the subject.

In some embodiments, rejuvenation of aged immune cells results in a reduction in the number of diseased cells or infectious agents in the subject. In some embodiments, senescent immune cells include senescent NK cells, senescent NKT cells, senescent T cells, senescent B cells, senescent monocytes, senescent macrophages, senescent neutrophils, senescent senescent basophils, senescent eosinophils, senescent Kupffer cells, and senescent microgial cells. In some embodiments, diseased cells include cancer cells, virus-infected cells, and intracellular bacteria-infected cells. In some embodiments, infectious agents include viruses, bacteria, fungi, and parasites.

Also provided herein are methods of using conjugates of common gamma chain cytokines and their cognate receptors and/or agonists that result in decreased activation of TGF-β receptors to rejuvenate the immune system. be done.

Methods of Improving Skin and/or Hair Texture and/or Appearance A method for improving skin and/or hair texture and/or appearance over a period of time (e.g., any of the periods described herein) is in need of improvement in skin and/or hair texture and/or appearance. A method of improving the texture and/or appearance of skin and/or hair in a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of one or more natural killer (NK) cell activators (e.g., Also provided herein are methods comprising administering an NK cell activator (any of the NK cell activators described or known in the art).

Improving skin and/or hair texture and/or appearance in a subject in need of improved skin and/or hair texture and/or appearance over a period of time (e.g., any of the periods described herein) A method of amelioration, wherein the subject is provided with a therapeutically effective amount of activated NK cells (e.g., any of the activated NK cells described herein or known in the art). Methods are also provided herein that include administering.

Some embodiments of these methods include obtaining resting NK cells and contacting the resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators, wherein Contacting results in the generation of activated NK cells that are then administered to the subject. In some examples of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some examples of these methods, the resting NK cells are haploidentical NK cells obtained from the subject. In some examples of these methods, the resting NK cells are allogeneic resting NK cells. In some examples of these methods, the resting NK cells are induced NK cells. In some examples of any of these methods, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

In some examples of these methods, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically defined liquid culture medium. Some examples of these methods include isolating activated NK cells (and administering a therapeutically effective amount of the activated NK cells to a subject, e.g., one of the subjects described herein). further administering to either). In some embodiments of these methods, the contacting step is carried out for a period of time from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of these methods, the methods provide an improvement in the subject's skin texture and/or appearance over a period of time (eg, any of the periods described herein).

In some embodiments of these methods, the method reduces the rate of wrinkle formation in the subject's skin over a period of time (e.g., any of the time periods described herein) (e.g., a reduction of at least 5%) , at least 10% reduced, at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced , at least 60% decreased, at least 65% decreased, at least 70% decreased, at least 75% decreased, at least 80% decreased, at least 85% decreased, at least 90% decreased, or at least 95% decreased, or about 5% decreased to about 99% % reduction (or any of the subranges of this range described herein) resulting in (e.g., the rate of wrinkle formation in a subject prior to treatment or the rate of wrinkle formation in a similar untreated subject compared to ).

In some embodiments of these methods, the methods result in an improvement in the subject's skin pigmentation over a period of time (eg, any of the periods of time described herein).

In some embodiments of these methods, the methods result in an improvement in the subject's skin texture over a period of time (eg, any of the periods of time described herein).

In some embodiments of these methods, the methods provide an improvement in the texture and/or appearance of the subject's hair over a period of time (eg, any of the periods described herein).

In some embodiments of these methods, the method reduces the rate of gray hair formation in a subject over a period of time (e.g., any of the time periods described herein) (e.g., at least a 5% reduction, at least 10% reduced, at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% to about 99% reduced (or any of the subranges of this range described herein) (e.g., compared to the rate of gray hair formation in a subject prior to treatment or the rate of gray hair formation in a similar untreated subject) do).

In some embodiments of these methods, the method reduces the number of gray hairs in the subject over a period of time (e.g., any of the time periods described herein) (e.g., at least a 5% reduction, at least 10 % reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduction, at least 45% reduction, at least 50% reduction, at least 55% reduction, at least 60 % reduction, at least 65% reduction, at least 70% reduction, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, or at least 95% reduction, or about 5% reduction to about 99% reduction ( or any of the subranges of this range described herein) (e.g., compared to the number of gray hairs in a subject before treatment or the rate of gray hair formation in a similar untreated subject) ).

In some embodiments of these methods, the methods reduce the hair loss rate (e.g., at least 5% reduction, at least 10% reduced, at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% reduced to about 99% reduced (or any of the subranges of this range described herein)) (eg, compared to the rate of hair loss in a subject prior to treatment or the rate of hair loss in a similar untreated subject).

In some embodiments of these methods, the methods result in an improvement in the texture of the subject's hair over a period of time (eg, any of the periods of time described herein).

In some embodiments of these methods, the methods reduce the number of senescent skin cells (e.g., by at least 5%) in the subject's skin over a period of time (e.g., any of the time periods described herein). reduced, at least 10% reduced, at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% to about 99% reduction (or any of the subranges of this range described herein) (e.g., the number of aging skin cells in a subject prior to treatment or aging skin in a similar subject not receiving treatment). compared to the number of cells).

In some embodiments of these methods, an improvement in the subject's skin texture and/or appearance over a period of time (e.g., any of the periods described herein) is determined by the presence, size, and shape, skin color and pigmentation, skin moisture, temperature, elasticity, and vascularity. .

In some embodiments of these methods, the improvement in the texture and/or appearance of the subject's hair over a period of time (e.g., any of the time periods described herein) is as described herein or It can be evaluated by any method known in the art.

In some embodiments of these methods, the time period is, for example, 1 month to 10 years, 1 month to 9 years, 1 month to 8 years, 1 month to 7 years, 1 month to 6 years, 1 month to 5 years. years, 1 month to 4 years, 1 month to 3 years, 1 month to 2 years, 1 month to 18 months, 1 month to 12 months, 1 month to 10 months, 1 month to 8 months, 1 month to 6 months, 1 month to 4 months, 1 month to 2 months, 1 month to 6 weeks, 6 weeks to 10 years, 6 weeks to 9 years, 6 weeks to 8 years, 6 weeks to 7 years, 6 weeks to 6 years, 6 weeks ~5 years, 6 weeks~4 years, 6 weeks~3 years, 6 weeks~2 years, 6 weeks~18 months, 6 weeks~12 months, 6 weeks~10 months, 6 weeks~8 months, 6 weeks~6 months, 6 weeks to 4 months, 6 weeks to 2 months, 2 months to 10 years, 2 months to 9 years, 2 months to 8 years, 2 months to 7 years, 2 months to 6 years, 2 months to 5 years, 2 months to 4 years, 2 months to 3 years, 2 months to 2 years, 2 months to 18 months, 2 months to 12 months, 2 months to 10 months, 2 months to 8 months,] 2 months to 6 months, 2 Months - 4 months, 4 months - 10 years, 4 months - 9 years, 4 months - 8 years, 4 months - 7 years, 4 months - 6 years, 4 months - 5 years, 4 months - 4 years, 4 months - 3 years, 4 months to 2 years, 4 months to 18 months, 4 months to 12 months, 4 months to 10 months, 4 months to 8 months, 4 months to 6 months, 6 months to 10 years, 6 months to 9 years , 6 months to 8 years, 6 months to 7 years, 6 months to 6 years, 6 months to 5 years, 6 months to 4 years, 6 months to 3 years, 6 months to 2 years, 6 months to 18 months, 6 Months - 12 months, 6 months - 10 months, 6 months - 8 months, 8 months - 10 years, 8 months - 9 years, 8 months - 8 years, 8 months - 7 years, 8 months - 6 years, 8 months - 5 years, 8 months to 4 years, 8 months to 3 years, 8 months to 2 years, multiple months to 18 months, 8 months to 12 months, 8 months to 10 months, 10 months to 10 years, 10 months to 9 years , 10 months to 8 years, 10 months to 7 years, 10 months to 6 years, 10 months to 5 years, 10 months to 4 years, 10 months to 3 years, 10 months to 2 years, 10 months to 18 months, 10 Months - 12 months, 12 months - 10 years, 12 months - 9 years, 12 months - 8 years, 12 months - 7 years, 12 months - 6 years, 12 months - 5 years, 12 months - 4 years, 12 months - 3 years, 12 months to 2 years, 12 months to 18 months, 18 months to 10 years, 18 months to 9 years, 18 months to 8 years, 18 months to 7 years, 18 months to 6 years, 18 months to 5 years , 18 months to 4 years, 18 months to 3 years, 18 months to 2 years, 2 years to 10 years, 2 years to 9 years, 2 years to 8 years, 2 years to 7 years, 2 years to 6 years, 2 Years to 5 years, 2 years to 4 years, 2 years to 3 years, 3 years to 10 years, 3 years to 9 years, 3 years to 8 years, 3 years to 7 years, 3 years to 6 years, 3 years and up 5 years, 3 years to 4 years, 4 years to 10 years, 4 years to 9 years, 4 years to 8 years, 4 years to 7 years, 4 years to 6 years, 4 years to 5 years, 5 years to 10 years , 5-9 years, 5-8 years, 5-7 years, 5-6 years, 6-10 years, 6-9 years, 6-8 years, 6-7 years, 7 years to 10 years, 7 years to 9 years, 7 years to 8 years, 8 years to 10 years, 8 years to 9 years, or 9 years to 10 years.

In some embodiments of these methods, the age of the subject is from about 30 to about 35, from about 35 to about 40, from about 40 to about 45, from about 45 to about 50, from about 50 to about 55, from about 55 to about 60, about 60 to about 65, about 65 to about 70, about 70 to about 75, about 75 to about 80, about 80 to about 85, about 85 to about 90, about 90 to about 95, about 95 to about 100, about 100 to about 105, about 105 to about 110, about 110 to about 115, or about 115 to about 120.

Method of Helping Treat Obesity in a Subject A method of helping treat obesity in a subject in need of help treating obesity for a period of time (e.g., any of the time periods 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) A method is provided herein comprising:

A method of helping treat obesity in a subject in need of help treating obesity for a period of time (e.g., any of the periods described herein) comprising administering to the subject a therapeutically effective amount of an active Also provided herein are methods comprising administering activated NK cells (e.g., any of the activated NK cells described herein or known in the art).

Some embodiments of these methods are obtaining resting NK cells and contacting the resting NK cells in vitro in a liquid culture medium comprising one or more resting NK cell activators, wherein Contacting results in the generation of activated NK cells that are then administered to the subject. In some examples of these methods, the resting NK cells are autologous NK cells obtained from the subject. In some examples of these methods, the resting NK cells are haploidentical NK cells obtained from the subject. In some examples of these methods, the resting NK cells are allogeneic resting NK cells. In some examples of these methods, the resting NK cells are induced NK cells. In some examples of any of these methods, the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

In some examples of these methods, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically defined liquid culture medium. Some examples of these methods include isolating activated NK cells (and administering a therapeutically effective amount of the activated NK cells to a subject, e.g., one of the subjects described herein). further administering to either). In some embodiments of these methods, the contacting step is carried out for a period of time from about 2 hours to about 20 days (or any subrange of this range described herein).

In some embodiments of these methods, the method reduces body weight (e.g., at least 5% loss, at least 10% loss) over a period of time (e.g., any of the time periods described herein) , at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced , at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% reduced to about 99% reduced (or this any of the subranges of this range described herein)) (eg, relative to the subject's weight prior to treatment).

In some embodiments of these methods, the method reduces a subject's body mass index (BMI) over a period of time (e.g., any of the time periods described herein) (e.g., a reduction of at least 5%) , at least 10% reduced, at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced , at least 60% decreased, at least 65% decreased, at least 70% decreased, at least 75% decreased, at least 80% decreased, at least 85% decreased, at least 90% decreased, or at least 95% decreased, or about 5% decreased to about 99% % reduction (or any of the subranges of this range described herein)) (eg, as compared to the subject's BMI prior to treatment).

In some embodiments of these methods, the method reduces the rate of progression from prediabetes to type 2 diabetes in a subject (e.g., at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction) , at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced , at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or from about 5% reduced to about 99% reduced (or subranges of this range described herein (e.g., compared to the rate of progression from prediabetes to type 2 diabetes in a subject before treatment or the rate of progression from prediabetes to type 2 diabetes in a similar untreated subject hand).

In some embodiments of these methods, the method reduces fasting serum glucose levels in the subject (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction) , at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced , at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or from about 5% reduced to about 99% reduced (or any subrange of this range described herein) )) (eg, compared to fasting serum glucose levels in the subject prior to treatment).

In some embodiments of these methods, the methods increase insulin sensitivity in the subject (e.g., at least 5% increase, at least 10% increase, at least 15% increase, at least 20% increase, at least 25% increase, at least 30% increase, % increase, at least 35% increase, at least 40% increase, at least 45% increase, at least 50% increase, at least 55% increase, at least 60% increase, at least 65% increase, at least 70% increase, at least 75% increase, at least 80 % increase, at least 85% increase, at least 90% increase, at least 95% increase, or at least 99% increase, or from about 10% increase to about 500% increase (or any subrange of this range described herein) either)) (compared to insulin sensitivity in the subject prior to treatment).

In some embodiments of these methods, the method reduces the severity of atherosclerosis in the subject (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduction, at least 80% reduction, at least 85% reduction, at least 90% reduction, or at least 95% reduction, or about 5% reduction to about 99% reduction (or any subrange of this range described herein) (e.g., compared to the severity of atherosclerosis in the subject prior to treatment).

In some embodiments of these methods, treating obesity in a subject over a period of time (e.g., any of the periods described herein) is, for example, body weight and/or body dimensions, total body fat, It can be assessed by any method described herein or known in the art, including systemic or regional obesity, and measuring body mass index (BMI).

In some embodiments of these methods, a subject's response to treatment can be monitored by determining fasting blood glucose levels or glucose tolerance according to standard techniques. In some embodiments of these methods, insulin sensitivity is measured as described herein, including Hyperinsulin Euglycemic Clamp and Intravenous Glucose Tolerance Test, Homeostasis Model Assessment (HOMA), and Quantitative Insulin Sensitivity Check Index (QUICKI). It can be measured using any method described or known in the art.

In some embodiments of these methods, the severity of atherosclerosis in a subject is determined by cardiac catheterization, Doppler ultrasonography, blood pressure comparison, MUGA/radionuclide angiography, thallium/myocardial perfusion scan, and computer It can be measured using any method described herein or known in the art, including tomography.

In some embodiments of these methods, the time period is from 1 month to 10 years (or any of the subranges of this range described herein).

In some embodiments of these methods, the subject's age range is from about 1 to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, from about 20 to about 25, from about 25 to about about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 50 to about 55, about 55 to about 60, about 60 to about 65, about 65 to about 70 , about 70 to about 75, about 75 to about 80, about 80 to about 85, about 85 to about 90, about 90 to about 95, about 95 to about 100, about 100 to about 105, about 105 to about 110, about 110 to about 115, or about 115 to about 120.

Additional Therapeutic Agents Some embodiments of any of the methods described herein include administering a therapeutically effective amount of one or more additional therapeutic agents to a subject (e.g., among the subjects described herein). any of the above). The one or more additional therapeutic agents can be administered to the subject at substantially the same time as the NK cell activator or activated NK cells (e.g., administered to the subject as a single formulation or as two or more formulations). obtain). In some embodiments, one or more additional therapeutic agents may be administered to the subject prior to administering the NK cell activator or activated NK cells. In some embodiments, one or more additional therapeutic agents may be administered to the subject after administering the NK cell activator or activated NK cells to the subject.

Non-limiting examples of additional therapeutic agents include anticancer agents, activating 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 agents include antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxycarbamide, methotrexate, 6-thioguanine, cladribine, nerarabine, pentostatin, or pemetrexed), plant alkaloids (e.g. vinblastine, vincristine, vindesine, camptothecin, 9-methoxycamptothecin, coronarysine, taxol, naucleaoral) , diprenylated indole alkaloids, montamine, shishkiniin, protoberberine, berberine, sanguinaline, chelerythrine, chelidonin, liriodenine, crivorine, β-carboline, antophine, tyrophorine, cryptotrepin, neocryptrepin, corinoline, sampangin, carbazole, clinamin, montanine , ellipticine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan, or acridone alkaloids), proteasome inhibitors (e.g., lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib (ONX- 0912), delanzomib (CEP-18770), epoxomicin, MG132, beta-hydroxy beta-methylbutyrate, bortezomib, carfilzomib, or ixazomib), antitumor antibiotics (e.g., doxorubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin, actinomycin, plicamycin, mitomycin, or bleomycin), histone deacetylase inhibitors (e.g., vorinostat, panobinostat, gibinostat, abexinostat, depsipeptide, entinostat, phenylbutyrate, valproic acid, trichostatin A, dacinostat, mosetinostat, prasinostat, nicotinamide, cambinol, tenovin 1, tenovin 6, sirtinol, licorinostat, tefinostat, cevetrin, xinostat, resminostat, tacedinaline, thidamide, or sericistat), tyrosine kinase inhibitors (e.g. , axitinib, dasatinib, encorafinib, erlotinib, imatinib, nilotinib, pazopanib, and sunitinib), and chemotherapeutic agents (e.g., all-trans retinoic acid, azacitidine, azathioprine, doxifluridine, epothilone, hydroxyurea, imatinib, teniposide, thioguanine, valrubicin, vemurafenib, and lenalidomide). Additional examples of chemotherapeutic agents include alkylating agents such as mechlorethamine, cyclophosphamide, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, altretamine, procarbazine, dacarbazine, temozolomide, carmustine, lumustine, Streptozocin, carboplatin, cisplatin, and oxaliplatin.

Non-limiting examples of activating receptor agonists include anti-CD16 antibodies (e.g., anti-CD16/CD30 bispecific monoclonal antibodies (BiMAbs)) and Fc-based fusion proteins that induce NK cell cytotoxicity. Any agonist for an activated receptor that activates and potentiates is included. Non-limiting examples of checkpoint inhibitors include anti-PD-1 antibodies (eg, MEDI0680), anti-PD-L1 antibodies (eg, BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316, anti-PD-L1/CTLA-4 bispecific antibody KN046, anti-PD-L1/TGFβRII fusion protein M7824, anti-PD-L1/TIM-3 bispecific anti-TIM3 antibody (e.g., TSR-022, Sym023, or MBG453), and anti-CTLA-4 antibody (e.g., AGEN1884, MK-1308, or anti-CTLA-4/OX40 dual specific antibody ATOR-1015). Non-limiting examples of agents for blocking HLA-specific inhibitory receptors include monalizumab (eg, anti-HLA-ENKG2A inhibitory receptor monoclonal antibody). Non-limiting examples of GSK3 inhibitors include Tideglusib or CHIR99021. Non-limiting examples of antibodies that can be used as additional therapeutic agents include anti-CD26 antibodies (e.g., YS110), anti-CD36 antibodies, and anti-CD36 antibodies that bind and activate Fc receptors (e.g., CD16) on NK cells. Any other antibody or antibody construct capable of In some embodiments, the additional therapeutic agent can be insulin or metformin.

Exemplary Methods Comprising Administration of One or More Common Gamma Chain Family Cytokine Receptor Activators A subject comprising administering a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators to the subject Provided herein are methods of killing naturally occurring and/or treatment-induced senescent cells in or reducing the number of senescent cells.

Also herein are methods of reducing spontaneous and/or treatment-induced accumulation of 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.

Also herein are methods of reducing the levels of markers of spontaneous 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. provided in the book. In some embodiments, the markers of spontaneous and/or treatment-induced senescent cells are p21 ^CIP1 p21 and CD26. Additional markers of spontaneous and/or treatment-induced senescent cells are described herein. Additional markers of spontaneous and/or therapy-induced senescent cells are known in the art.

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

One or more senescence-associated secretory expressions derived from 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 a type (SASP) factor. In some embodiments, the senescent cells express inflammatory properties and the inflammatory properties are SASP factors. In some embodiments, senescence-associated secretory phenotype (SASP) factors include inflammatory cytokines (eg, IL-1α, IL-1β, IL-6, IL-8, TNF-α), growth factors (eg, , 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 (eg, MMP-3, MMP-9). In some embodiments, the method reduces the expression level and/or activity of one or more (e.g., 2, 3, 4, or 5) of senescence-associated secretory phenotype (SASP) factors Let In some embodiments, the SASP factor expression level or activity is determined using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the SASP factor expression level or activity is determined using immunoblotting.

In some embodiments of any of the methods described herein, the subject has an age-related disease (e.g., an exemplary type of aging described herein or known in the art). previously diagnosed or identified as having an age-related disease or condition) or an inflammatory disease (e.g., exemplary types of age-related diseases or conditions described herein or known in the art) It is

In some embodiments, the age-related disease is inflammatory senescence-related.

In some embodiments, the age-related disease is cancer (eg, any of the exemplary types of cancer described herein or known in the art).

In some embodiments of any of the methods described herein, the inflammatory disease is 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-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, nonalcoholic steatohepatitis, mood disorders, and cancer treatment-related cognition selected from the group consisting of disorders;

In some examples of these methods, the treatment-induced senescent cells are chemotherapy-induced senescent cells.

In some embodiments of these methods, administering is a naturally occurring target tissue of the subject (e.g., any of the exemplary types of target tissue described herein or known in the art). and/or a reduction in the number of treatment-induced senescent cells (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least a 90% reduction, or at least a 95% reduction, or from about a 5% reduction to about a 99% reduction (or any of the subranges of this range described herein) (e.g., pre-treatment subjects) (compared to the number of spontaneous and/or treatment-induced senescent cells in the target tissue in the 2000s).

In some embodiments of these methods, administering (e.g., for any of the time periods described herein) reduces the accumulation of spontaneous and/or treatment-induced senescent cells in the subject (e.g., at least 5% reduced, at least 10% reduced, at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% reduced to about a 99% reduction (or any of the subranges of this range described herein) (e.g., spontaneous and/or treatment-induced accumulation of senescent cells in the subject prior to treatment, or treatment (compared to spontaneous and/or treatment-induced accumulation of senescent cells in similar subjects not receiving treatment).

In some embodiments of these methods, administration reduces the level of one or more (e.g., two, three, or four) markers of spontaneous and/or treatment-induced senescent cells in the subject ( For example, at least 5% decreased, at least 10% decreased, at least 15% decreased, at least 20% decreased, at least 25% decreased, at least 30% decreased, at least 35% decreased, at least 40% decreased, at least 45% decreased, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% to about 99% reduction (or any of the subranges of this range described herein) (e.g., one of the naturally occurring and/or treatment-induced senescent cells in the subject prior to treatment). compared to the levels of one or more markers).

As used herein, "spontaneous senescent cells" are senescent cells that are produced as a result of normal aging or inflammatory processes. Spontaneously occurring senescent cells can accumulate over time in various tissues and organs of an individual. Spontaneous senescent cells can be any of the exemplary types of senescent cells described herein that are not induced by therapeutic treatment (eg, chemotherapy or radiation).

As used herein, "treatment-induced senescent cells" are senescent cells produced as a result of therapeutic treatment (eg, chemotherapy or radiation).

Common Gamma Chain Family Cytokine Receptor Activators Methods are provided herein that involve 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 (eg, any of the exemplary single-chain chimeric polypeptides described herein), a multi-chain Chimeric polypeptides (eg, any of the exemplary multi-chain chimeric polypeptides described herein), soluble IL-15 or IL-15 agonists (eg, soluble IL-15 or IL-15 described herein) 15 agonists), soluble IL-2 or IL-2 agonists (eg, any of the soluble IL-2 or IL-2 agonists described herein), common gamma chain family cytokines (or functional fragments thereof) ), as well as antibodies (or antibody fragments) or functional fragments thereof that specifically bind to common gamma chain family cytokines, antibodies or antigen-binding antibody fragments that specifically bind to cytokines of the common gamma chain family.

Exemplary Single Chain Chimeric Polypeptides Non-limiting examples of common gamma chain family cytokine receptor activators include (i) a first target binding domain, (ii) a soluble tissue factor domain (e.g., or any of the exemplary target binding domains known in the art), and (iii) a second target binding domain, the first target binding domain and the second target binding domain is a soluble common gamma chain family cytokine, an agonist 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 common gamma chain family cytokines.

Some embodiments of any of the single-chain chimeric polypeptides described herein have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (eg, 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 first target binding domain (e.g., an exemplary any of the target binding domains), 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 One or more of the additional target binding domains (eg, any of the exemplary target binding domains described herein or known in the art) are soluble common gamma chain family cytokines. 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 are soluble common gamma chain family cytokine receptors (e.g., IL-2, IL-4, IL-7 , IL-9, IL-15, and IL-21).

In some embodiments of any of the single chain chimeric polypeptides described herein, the first target binding domain (e.g., an exemplary any of the target binding domains), 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 one or more of the additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are specific for a common gamma chain family cytokine receptor It is an agonist antigen-binding domain that specifically binds. 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. mentioned.

Multichain Chimeric Polypeptides Non-limiting examples of common gamma chain family cytokine receptor activators include (a) (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a pair of a first chimeric polypeptide comprising a first of the affinity domains; and (b) a second chimeric polypeptide comprising (i) a second of the pair of affinity domains and (ii) a second target binding domain. and wherein one or both of the first target binding domain and the second target binding domain are a soluble common gamma chain family cytokine, 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 has one or more (e.g., two, three, four, five, six, seven, 8, 9, or 10) additional target binding domains (eg, 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., exemplary targets described herein or known in the art) binding domain), 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 (e.g., any of the exemplary target binding domains described herein or known in the art) of (e.g., two, three, four, 5, 6, 7, 8, 9, or 10) are soluble common gamma chain family cytokines. 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, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain), 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 (e.g., any of the exemplary target binding domains described herein or known in the art) of are specific for a common gamma chain family cytokine receptor is an agonist antigen-binding domain that binds to 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. mentioned.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., exemplary targets described herein or known in the art) binding domain), 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 (e.g., any of the exemplary target binding domains described herein or known in the art) of (e.g., two, three, four, 5, 6, 7, 8, 9, or 10) are soluble common gamma chain family cytokine receptors.

In some embodiments of the multi-chain chimeric polypeptides described herein, the first domain or the second domain of the pair of affinity domains is a soluble common gamma chain family cytokine or a common gamma chain family cytokine receptor. It is an antigen-binding domain that specifically binds to the body.

ヒト可溶性ＩＬ－９（配列番号３３６）

Soluble Common Gamma Chain Family Cytokine In some embodiments of any of the single- or multi-chain chimeric polypeptides described herein, one of the first target-binding domain and the second target-binding domain One or both may be a soluble common gamma chain family cytokine. In some embodiments, the common gamma chain family cytokine receptor activator can 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 soluble IL-2, soluble IL-7, soluble IL-15, and soluble IL-21 sequences are provided herein. Non-limiting examples of soluble IL-4 and IL-9 sequences are provided 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 the antigen-binding domain. 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 are each antigen binding domains. In some embodiments of any of the single- or multi-chain chimeric polypeptides described herein, the antigen binding domain is a scFv or single domain antibody (eg, Va _H H or V _NAR domains) contains or is

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 share An agonist antigen-binding domain that specifically binds to gamma chain family cytokine receptors. In some examples, the agonist antigen binding domain (eg, any of the antigen binding domains described herein) is IL-2, IL-4, IL-7, IL-9, IL-15 or It can specifically bind to the receptor for IL-21.

Each antigen-binding domain present in any of the single- or multi-chain chimeric polypeptides described herein is independently selected from the group consisting of VHH domains, VNAR domains, and scFv. In some embodiments, any of the antigen binding domains described herein are BiTe, (scFv) ₂ , Nanobody, Nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH- CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. Additional examples of antigen binding domains that can be used in either single-chain chimeric polypeptides or multi-chain chimeric polypeptides are known in the art.

In some embodiments, each of the antigen binding domains within the single-chain or multi-chain chimeric polypeptides described herein are both VHH domains, or at least one antigen binding domain is a VHH domain . In some embodiments, each of the antigen binding domains within the single or multi-chain chimeric polypeptides described herein are both VNAR domains, or at least one antigen binding domain is a VNAR domain. . In some embodiments, each of the antigen binding domains of the single or multi-chain chimeric polypeptides described herein are both scFv domains, 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 are assembled (e.g., non-covalently assembled) to form an antigen described herein. Any of the binding domains, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), VHH-scAb, VHH-Fab, double scFab, F ( ab′) ₂ , diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange , SEEDbody, LUZ-Y, Fcab, κλ-body, 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-CH3, Triple body, Mino antibody, Minibody, TriBi minibody, scFv-CH3KIH, Fab-scFv, F(ab') ₂ -scFv ₂ , scFv-KIH, Fab- scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv1- PEG-scFv ₂ can be formed. For a description of these elements, see, for example, Spiess et al. , Mol. Immunol. 67:95-106, 2015. Non-limiting examples of antigen-binding fragments of antibodies include Fv, Fab, F(ab') ₂ , and Fab' fragments. Additional examples of antigen-binding fragments of antibodies include antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., human or humanized IgG, e.g., human or humanized IgG1, IgG2 , IgG3, or IgG4), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., human or humanized IgA, e.g., antigen-binding fragments of human or humanized IgA1 or IgA2) , an antigen-binding fragment of IgD (e.g., an antigen-binding fragment of human or humanized IgD), an antigen-binding fragment of IgE (e.g., an antigen-binding fragment of human or humanized IgE), or an antigen-binding fragment of IgM (e.g., human or antigen-binding fragment of 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 has a unique high-affinity binding IL-15Rα chain that confers receptor specificity for IL-15, and a common IL-15Rβ and γ chain shared with IL-2 (also known as IL-2Rβ/γ). It functions through a trimeric IL-15 receptor complex consisting of

In some embodiments, the soluble IL-15 is SEQ ID NO: 82 and at least 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% %) are identical. In some embodiments, the soluble IL-15 is recombinant soluble human IL-15. In some embodiments, the soluble IL-15 is mutant IL-15 having one or more amino acid substitutions compared to wild-type IL-15 (eg, SEQ ID NO:82). Mutant IL-15 can include, for example, a D8N or D8A amino acid substitution compared to wild-type IL-15. In some embodiments, soluble IL-15 can be conjugated to polymers (eg, Miyazaki et al., Proceed. Annual Meeting AACR, 2019, Abstract 3265).

Some examples of IL-15 agonists described herein may include complexes of IL-15 with all or part of the soluble IL-15 receptor (IL-15R). Complexes of IL-15 with all or part of soluble IL-15R may have increased half-life and/or increased potency compared to free IL-15. In some embodiments, an IL-15 agonist described herein further comprises an Fc domain (eg, any of the exemplary Fc domains described herein).

In some embodiments, the portion of soluble IL-15R is IL-15Rα. For example, IL-15 can bind to an IL-15Rα-Fc fusion to form an IL-15:IL-15Rα-Fc complex (see, eg, Stoklasek et al., J. Immunology 177:6072 -80, 2006, Dubios et al., J. Immunol.180:2099-106, 2008, Epardaud et al., Cancer Res.68:2972-83, 2008, Rubinstein et al., Proc.Natl.Acad.Sci. U.S.A. 103:9166-71, 2006). In some embodiments, soluble IL-15 and IL-15Rα form a heterodimer (see, eg, Colon et al., Cancer Res. 79 (13 Supplement): CT082, July 1, 2019).

In some embodiments, the portion of soluble IL-15R is a portion of IL-15Rα (eg, the sushi domain of IL-15Rα).

IL-15 in the complex can be wild-type IL-15 or mutated IL-15. For example, a mutated IL-15 containing the N72D mutation can be used to form a complex with all or part of a soluble IL-15R (eg, the sushi domain of IL-15Rα). In some embodiments, the complex is ALT-803 and comprises the human IL-15 variant IL-15N72D complexed with an IL-15Rα sushi-Fc fusion (see, eg, Zhu et al., J. Immunol.183(6):3598-607, 2009).

Non-limiting examples of IL-15 agonists include ALT-803/N-803 (Altor Bioscience/ImmunityBio), BNZ-1 (Bioniz Therapeutics), NIZ985 (Novartis), RTX-212 (Rubis Therapeutics), AM0 015 ( rhIL-15) (Lilly), IGM-7354 (IGM), XmAb24306 (Roche/Xencor), KD033 (srKD033) (Kadmon), OXS-C3550 (GT Biopharma), and NKTR-255 (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 centrally involved in immune tolerance and immune activation through its effects on CD4 ⁺ T regulatory cells and cytotoxic effector lymphocytes such as CD8 ⁺ T cells and NK cells. IL-2 acts on cells expressing either the dimeric IL-2 receptor (IL-2R), which consists of the IL-2Rβ and γ chains, or the trimeric αβγ receptor (IL-2Rαβγ), The trimeric receptor exhibits a 10-100 fold higher affinity for IL-2 compared to the dimeric IL-2R. CD4 ⁺ regulatory T cells are characterized by strong constitutive expression of IL-2Rα. This allows the cells to express IL-2Rαβγ and thereby use lower levels of IL-2. Dimeric IL-2R is most prominent on antigen-experienced (memory) CD8 ⁺ T cells and NK cells. Thus, high levels of IL-2, in addition to activating Treg cells, potently stimulate CD8 ⁺ T cells and NK cells.

In some embodiments, the soluble IL-2 is at least 90% (eg, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:78. be. In some embodiments, soluble IL-2 is recombinant human IL-2. Soluble IL-2 can be an IL-2 variant. For example, an IL-2 variant may bind IL-2Rβ more effectively (eg, at least 50, 100, 150 or 200 times more effectively) than IL-2Rα. An exemplary IL-2 variant is MDNA109 (see, eg, Rafei et al., J. Clin. Oncol. 37 (15Suppl.), 2019). Disabling bindings. For example, residues F42, Y45, and L72 involved in CD25 binding can be mutated (see, eg, Klein et al., Oncoimmunology 6(3): e1277306, 2017).

In some embodiments, the IL-2 agonist is pegylated IL-2, which has limited binding to the IL2Rα subunit and preferentially binds dimeric IL2Rβγ (eg, Bentebibel et al., Cancer Discov .9(6):711-721, 2019).

Some examples of IL-2 agonists described herein are fusion proteins that include IL-2. In some embodiments, the fusion protein comprises IL-2 or a variant thereof linked to all or part of a soluble IL-2R. In some embodiments, a portion of the soluble IL-2R is IL-2Rα (see, eg, Vaishampayan et al., J. Clin. Oncol. 35(15Suppl.), 2017). A fusion protein can, for example, selectively activate dimeric IL-2Rβγ. Further examples of IL-2 fusion proteins include those fused to toxins (eg, diphtheria toxin).

In some embodiments, the fusion protein comprises IL-2 or a variant thereof (eg, any of the IL-2 variants described herein) linked to an antibody (eg, monoclonal antibody or scFv). include. Non-limiting examples of antibodies that can be linked to IL-2 or variants thereof include human monoclonal antibodies against fibroblast activation protein-alpha (FAP) (eg, Soerensen et al., J. Clin. Oncol. 36). , No. 15 Suppl.), anti-CD20 monoclonal antibodies (see, e.g., Lansigan et al., Blood 128(22):620, 2016), scFv against the A1 domain of tenascin-C (e.g., Catania et al., Cell Adh. Migr. 9(1-2):14-21, 2015), and anti-CEA antibodies (see, eg, Klein et al., Oncoimmunol. 6(3): e1277306, 2017).

Additional examples of IL-2 agonists include proleukin (Clinigen), plumoleukin (Immunservice), NKTR-214 (Nektar Therapeutics), DI-Leu16-IL2 (Alopexx/Provenance Biopharmaceuticals), RG7461 (Roche ), Teleukin (Philogen ), ALT-801803 (Altor Bioscience), ALT-801 (Altor Bioscience), ALKS4230 (Alkermes), Sergutuzumab amnaleukin (RG7813) (Roche), Camidan lumabutecillin (ADC Therapeutics/Genbma b), NHS - IL2-LT/EMD521873 (Merck KGaA), NIZ985 (Novartis), MDNA109 (Medicenna Therapeutics), Angeloxin (Angelica Therapeutics), PB101 (Pivotal Biosciences), anti-IL-2 program (Xo ma), NKTR-255 (Nektar Therapeutics ), NKTR-358/LY3471851 (Nektar Therapeutics)/Lilly), CYP0150 (Cytunepharma), NL-201 (Neoleukin), THOR-809 (Sanofi/Synthorx), BNT151/153 (BioNTech) , transcon IL-2β/γ (Ascendis Pharma), ILT-101 (Servier)/ILT-101) and AM0015 (Lilly). Additional examples of IL-2 agonists are known in the art.

Conjugates of Common Gamma Chain Family Cytokines with Antibodies or Antibody Fragments Non-limiting examples of common gamma chain family cytokine receptor activators include common gamma chain family cytokines (e.g., common gamma chain family cytokine) and an antibody or antigen-binding antibody fragment that specifically binds to a 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 enhances the activity of the common gamma chain family cytokine and CD8 ⁺ Resulting in proliferation of T cells and/or NK cells. In some embodiments, the conjugate has a longer half-life in circulation than the free common gamma chain family cytokine.

In some embodiments, a conjugate may comprise soluble IL-2 (eg, recombinant soluble human IL-2) or 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 antibodies S4B6, JES6-5, or MAB602, respectively ( See, eg, Tomala et al., J. Immunol.183:4904-4912, 2009 and Boyman et al., Science 311, 2006).

In some embodiments, a conjugate may comprise soluble IL-4 (eg, 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, for example, Sato et al. , J. Immunol. 150:2717-2723, 1993, and Finkelman et al. , J. Immunol. 151:1235-1244, 1993.

In some embodiments, a conjugate may comprise soluble IL-7 (eg, 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, for example, Finkelman et al. , J. Immunol. 151:1235-1244, 1993, and Boyman et al. , J. Immunol. 180:7265-75, 2008.

In some examples of conjugates, the common gamma chain family cytokine (or functional fragment thereof) and the antibody (or antigen-binding antibody fragment thereof) can be administered separately, wherein the common gamma chain family cytokine and the antibody or antigen Complexes with bound antibody fragments can 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 Administration of One or More Agents That Result in Decreased Activation of TGF-β Receptors Subject to a therapeutically effective amount of one or more agents that result in decreased activation of TGF-β receptors Provided herein are methods of killing naturally occurring and/or treatment-induced senescent cells or reducing the number of senescent cells in a subject comprising administering to.

Also a method of reducing spontaneous 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. provided herein.

Reducing levels of spontaneous and/or treatment-induced markers 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-beta receptors Methods are also provided herein. In some embodiments, the markers of spontaneous and/or treatment-induced senescent cells are p21 ^CIP1 p21 and CD26. Additional markers of spontaneous and/or treatment-induced senescent cells are described herein. Additional markers of spontaneous and/or therapy-induced senescent cells are known in the art.

Also a method of reducing spontaneous and/or treatment-induced senescent cell activity in a subject comprising administering to the subject a therapeutically effective amount of one or more agents that result in reduced activation of TGF-beta receptors. provided herein.

one or more treatments derived from 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 decreased activation of TGF-β receptors. Also provided herein are methods of reducing the level and/or activity of SASP factors. In some embodiments, the senescent cells express inflammatory properties and the inflammatory properties are SASP factors. In some embodiments, SASP factors include inflammatory cytokines (eg, IL-1α, IL-1β, IL-6, IL-8, TNF-α), growth factors (eg, 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), but not limited to. In some embodiments, the method reduces the expression level or activity of one or more of the SASP factors. In some embodiments, the SASP factor expression level or activity is determined using an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the SASP factor expression level or activity is determined using immunoblotting.

In some embodiments of any of the methods described herein, the subject has an age-related disease (e.g., an exemplary type of aging described herein or known in the art). previously diagnosed or identified as having an age-related disease or condition) or an inflammatory disease (e.g., exemplary types of age-related diseases or conditions described herein or known in the art) It is

In some embodiments, the age-related disease is inflammatory senescence-related.

In some embodiments, the age-related disease is cancer (eg, any of the exemplary types of cancer described herein or known in the art).

In some embodiments of any of the methods described herein, the inflammatory disease is 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-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, nonalcoholic steatohepatitis, mood disorders, and cancer treatment-related cognition selected from the group consisting of disorders;

In some examples of these methods, the treatment-induced senescent cells are chemotherapy-induced senescent cells.

In some embodiments of these methods, administering is a naturally occurring target tissue of the subject (e.g., any of the exemplary types of target tissue described herein or known in the art). and/or a reduction in the number of treatment-induced senescent cells (e.g., at least 5% reduction, at least 10% reduction, at least 15% reduction, at least 20% reduction, at least 25% reduction, at least 30% reduction, at least 35% reduction, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least a 90% reduction, or at least a 95% reduction, or from about a 5% reduction to about a 99% reduction (or any of the subranges of this range described herein) (e.g., pre-treatment subjects) (compared to the number of spontaneous and/or treatment-induced senescent cells in the target tissue in 20 days).

In some embodiments of these methods, administering (e.g., for any of the time periods described herein) reduces the accumulation of spontaneous and/or treatment-induced senescent cells in the subject (e.g., at least 5% reduced, at least 10% reduced, at least 15% reduced, at least 20% reduced, at least 25% reduced, at least 30% reduced, at least 35% reduced, at least 40% reduced, at least 45% reduced, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% reduced to about a 99% reduction (or any of the subranges of this range described herein) (e.g., spontaneous and/or treatment-induced accumulation of senescent cells in the subject prior to treatment, or treatment (compared to spontaneous and/or treatment-induced accumulation of senescent cells in similar subjects not receiving treatment).

In some embodiments of these methods, administering reduces the levels of one or more (e.g., 2, 3, or 4) markers of spontaneous and/or treatment-induced senescent cells in the subject ( For example, at least 5% decreased, at least 10% decreased, at least 15% decreased, at least 20% decreased, at least 25% decreased, at least 30% decreased, at least 35% decreased, at least 40% decreased, at least 45% decreased, at least 50% reduced, at least 55% reduced, at least 60% reduced, at least 65% reduced, at least 70% reduced, at least 75% reduced, at least 80% reduced, at least 85% reduced, at least 90% reduced, or at least 95% reduced, or about 5% to about 99% reduction (or any of the subranges of this range described herein) (e.g., one of the naturally occurring and/or treatment-induced senescent cells in the subject prior to treatment). compared to the levels of one or more markers).

In some embodiments, the TGF-beta receptor is TGF-beta receptor II (TGF-betaRII).

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 decreased TGF-beta receptor activation is a soluble TGF-beta receptor, an extracellular domain of a TGF-beta receptor, An antibody that specifically binds to TGF-beta, an antagonistic antibody that binds to a TGF-beta receptor, an agent that binds to LAP, or an agent that binds to the TGF-beta/LAP complex. In some embodiments, the one or more agents that result in decreased activation of the TGF-beta receptor are those of the TGF-beta receptor through binding to LAP or to the TGF-beta/LAP complex. Decrease activation. Non-limiting examples of agents that result in decreased activation of TGF-β receptors are described below.

Agents That Result in Decreased Activation of TGF-β Receptors Methods are provided herein that involve the use or administration of one or more agents that result in decreased activation of TGF-β receptors. In some embodiments, the agent that results in decreased TGF-β receptor activation is a single-chain chimeric polypeptide (eg, one of the exemplary single-chain chimeric polypeptides described herein). any), a multi-chain chimeric polypeptide (eg, any of the exemplary multi-chain chimeric polypeptides described herein), a soluble TGF-β receptor, the extracellular domain of a TGF-β receptor, An antibody (or antibody fragment) that specifically binds to TGF-beta, an antagonistic antibody that binds to a TGF-beta receptor, an agent that binds LAP, or an agent that binds to the TGF-beta/LAP complex.

Exemplary Single Chain Chimeric Polypeptides Non-limiting examples of agents that result in decreased activation of the TGF-β receptor include (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; One or both of the one target-binding domain and the second target-binding domain specifically bind to a ligand of the TGF-β receptor, or the first target-binding domain and the second target-binding domain One or both of them are antagonist antigen binding domains that specifically bind to the 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 have one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (eg, 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 first target binding domain (e.g., an exemplary any of the target binding domains) 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 - Beta receptors. Non-limiting examples of soluble TGF-β receptors include soluble TGFβRI, soluble TGFβRII, soluble TGFβRIII, and soluble endoglin. Non-limiting sequences of exemplary soluble TGFβRII are described herein.

Exemplary Multichain Chimeric Polypeptides Non-limiting examples of agents that result in decreased TGF-β receptor activation include (a) (i) a first target binding domain, (ii) a soluble tissue factor domain and (iii) a first chimeric polypeptide comprising a first of the pair of affinity domains, and (b) (i) a second of the pair of affinity domains, and (ii) a second target a second chimeric polypeptide comprising a binding domain, wherein one or both of the first target-binding domain and the second target-binding domain are a ligand of a TGF-β receptor or one or both of the first and second target binding domains is an antagonist antigen binding domain that specifically binds to the TGF-β receptor.

In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide has one or more (e.g., two, three, four, five, six, seven, 8, 9, or 10) additional target binding domains (eg, 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 comprises one or more (e.g., two, three, four, five, six, seven, 8, 9, or 10) additional target binding domains (eg, 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., exemplary targets described herein or known in the art) binding domain) and/or the second target binding domain (eg, 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-β receptors include soluble TGFβRI, soluble TGFβRII, soluble TGFβRIII, and soluble endoglin.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the pair of affinity domains are a sushi domain derived from the alpha chain of the human IL-15 receptor (IL15Rα) and a soluble IL-15. 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 comprises mutations to reduce or eliminate IL-15 activity.

In some embodiments of any of the multi-chain chimeric polypeptides described herein, the pair of affinity domains are barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments. , and a SNARE module based on the interaction of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments of any of the multichain chimeric polypeptides described herein, the first domain or the second domain of the pair of affinity domains is a soluble common gamma chain family cytokine, or a common An antigen binding domain that specifically binds to gamma chain family cytokine receptors.

Non-limiting examples of multi-chain chimeric polypeptides that are agents that result in decreased activation of TGF-β receptors are described in "Exemplary Multi-Chain Chimeric Polypeptides—Types B, G, I, K, L, M, N, O, and P” in the subsections herein.

Soluble TGF-beta Receptor In some embodiments, the one or more agents that result in decreased activation of the TGF-beta receptor is a soluble TGF-beta receptor. In some embodiments, the one or more agents that result in decreased TGF-beta receptor activation are soluble TGF-beta receptors. Non-limiting examples of soluble TGF-β receptors include soluble TGFβRI, soluble TGFβRII, soluble TGFβRIII, and soluble endoglin.

In some embodiments, the TGF-beta receptor is TGF-beta receptor II (TGFβRII). In some embodiments, the TGFβ receptor is TGFβRIII.

The type I receptor, TGFβRI, is a membrane bound serine/threonine kinase that requires the presence of TGFβRII to bind TGF-β. The type II receptor, TGFβRII, is a membrane-associated serine/threonine kinase that binds TGF-β1 and TGF-β3 with high affinity and TGF-β2 with much lower affinity. In some embodiments, signaling requires the cytoplasmic domains of both TGFβRI and TGFβRII. The type III receptor, TGF-βRIII, is a proteoglycan that exists in membrane-bound and soluble forms and binds TGF-β1, TGF-β2, and TGF-β3, but is not involved in signal transduction. . Non-limiting examples of sequences for soluble TGFβRII 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 and second target binding domains It is an antigen-binding domain. 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 are each antigen binding domains. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises a scFv or single domain antibody (e.g., VHH or VNAR domains) , or it is.

In some embodiments of any of the single- or multi-chain chimeric polypeptides described herein, one or both of the first and second target binding domains are TGF - An antagonist antigen binding domain that specifically binds to β. In some examples, the antagonist antigen binding domain (e.g., any of the antigen binding domains described herein) specifically binds soluble TGFβRI, soluble TGFβRII, soluble TGFβRIII, or soluble endoglin. can be done.

In some embodiments, any of the antigen binding domains described herein are BiTe, (scFv) ₂ , Nanobody, Nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH- CL-scFv, HSAbody, scDiabody-HSA, or tandem-scFv. Additional examples of antigen binding domains that can be used in either single-chain chimeric polypeptides or multi-chain chimeric polypeptides are known in the art.

In some embodiments, two or more polypeptides present in a single-chain or multi-chain chimeric polypeptide are assembled (e.g., non-covalently assembled) to form an antigen described herein. Any of the binding domains, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), VHH-scAb, VHH-Fab, double scFab, F ( ab′) ₂ , diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange , SEEDbody, LUZ-Y, Fcab, κλ-body, 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-CH3, Triple body, Mino antibody, Minibody, TriBi minibody, scFv-CH3KIH, Fab-scFv, F(ab') ₂ -scFv ₂ , scFv-KIH, Fab- scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv1- PEG-scFv ₂ can be formed. For a description of these elements, see, for example, Spiess et al. , Mol. Immunol. 67:95-106, 2015.

Non-limiting examples of antigen-binding fragments of antibodies include Fv, Fab, F(ab') ₂ , and Fab' fragments. Additional examples of antigen-binding fragments of antibodies include antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., human or humanized IgG, e.g., human or humanized IgG1, IgG2 , IgG3, or IgG4), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., human or humanized IgA, e.g., antigen-binding fragments of human or humanized IgA1 or IgA2) , an antigen-binding fragment of IgD (e.g., an antigen-binding fragment of human or humanized IgD), an antigen-binding fragment of IgE (e.g., an antigen-binding fragment of human or humanized IgE), or an antigen-binding fragment of IgM (e.g., human or antigen-binding fragment of humanized IgM).

Agents that Bind Latent Associated Peptides (LAPs) Non-limiting examples of agents that bind latent associated peptides (LAPs) are TGF-β1, thrombospondin-1 (TSP-1), integrin αvβ6, or KRFK is a peptide. In some embodiments, LAP binds TGF-β1 to form a latent complex, and LAP is presumed to function as a sequestering agent of active TGF-β1. In some embodiments, LAP of a potential TGF-β complex also interacts with thrombospondin-1 (TSP-1) as part of a biologically active complex. Formation of the TSP-1/LAP complex involves the activation sequence of TPS-1 (KRFK) and a sequence near the amino terminus of LAP conserved in TGF-β1-5 (LSKL). The interaction of LAP with TSP-1 via the LSKL and KRFK sequences was thrombospondin-mediated, as LSKL peptides can competitively inhibit potential TGF-β activation by TSP-1 or KRFK-containing peptides. Important for potential TGF-β activation. In some embodiments, integrin αvβ6 has a high affinity for TGF-β1 LAP and has been shown to be involved in activation of the TGF-β1 latent complex.

Agents that bind to the TGF-β/LAP complex A non-limiting example of an agent that binds to the TGF-β/LAP complex is the latent TGF-β binding protein (LTBP). In some embodiments, a latent TGF-beta binding protein (LTBP) binds to the TFG-beta/LAP complex to form a larger complex called the large latent complex (LLC). In some embodiments, LTBPs include LTBP-1, LTBP-2, LTBP-3 and LTBP-4. In some embodiments, LTBP-1 forms a disulfide-bonded complex with a TGFβ propeptide (eg, LAP) within the endoplasmic reticulum. In some embodiments, LTBP-4 binds only to TGF-β1, thus mutations in LTBP-4 lead to TGF-β-related complications that are primarily tissue-specific in which TGF-β1 is involved. there is a possibility.

Methods of Administration Some embodiments of the methods described herein include administering one or more agents one or more times (e.g., three or more times, four 1 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more) doses to the subject. In some embodiments of these methods, any two sequential doses of the two or more doses are spaced about one week to about one year apart (eg, about one week to about 11 months, about one week ~ about 10 months, about 1 week to about 9 months, about 1 week to about 8 months, about 1 week to about 7 months, about 1 week to about 6 months, about 1 week to about 5 months, about 1 week to about 4 months, about 1 week to about 3 months, about 1 week to about 2 months, about 1 week to about 1 month, about 1 week to about 3 weeks, about 1 week to about 2 weeks, about 2 weeks to about 12 months , about 2 weeks to about 11 months, about 2 weeks to about 10 months, about 2 weeks to about 9 months, about 2 weeks to about 8 months, about 2 weeks to about 7 months, about 2 weeks to about 6 months, about 2 weeks to about 5 months, about 2 weeks to about 4 months, about 2 weeks to about 3 months, about 2 weeks to about 2 months, about 2 weeks to about 1 month, about 2 weeks to about 3 weeks, about 3 weeks ~ about 12 months, about 3 weeks to about 11 months, about 3 weeks to about 10 months, about 3 weeks to about 9 months, about 3 weeks to about 8 months, about 3 weeks to about 7 months, about 3 weeks to about 6 months, about 3 weeks to about 5 months, about 3 weeks to about 4 months, about 3 weeks to about 3 months, about 3 weeks to about 2 months, about 3 weeks to about 1 month, about 1 month to about 12 months , about 1 month to about 11 months, about 1 month to about 10 months, about 1 month to about 9 months, about 1 month to about 8 months, about 1 month to about 7 months, about 1 month to about 6 months, about 1 month to about 5 months, about 1 month to about 4 months, about 1 month to about 3 months, about 1 month to about 2 months, about 2 months to about 12 months, about 2 months to about 11 months, about 2 months ~ about 10 months, about 2 months to about 9 months, about 2 months to about 8 months, about 2 months to about 7 months, about 2 months to about 6 months, about 2 months to about 5 months, about 2 months to about 4 months, about 2 months to about 3 months, about 3 months to about 12 months, about 3 months to about 11 months, about 3 months to about 10 months, about 3 months to about 9 months, about 3 months to about 8 months , about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 4 months to about 12 months, about 4 months to about 11 months, about 4 months to about 10 months, about 4 months to about 9 months, about 4 months to about 8 months, about 4 months to about 7 months, about 4 months to about 6 months, about 4 months to about 5 months, about 4 months ~ about 4 months, about 5 months to about 12 months, about 5 months to about 11 months, about 5 months to about 10 months, about 5 months to about 9 months, about 5 months to about 8 months, about 5 months to about 7 months, about 5 months to about 6 months, about 6 months to about 12 months, about 6 months to about 11 months, about 6 months to about 10 months, about 6 months to about 9 months, about 6 months to about 8 months , about 6 months to about 7 months, about 7 months to about 12 months, about 7 months to about 11 months, about 7 months to about 10 months, about 7 months to about 9 months, about 7 months to about 8 months, about 8 months to about 12 months, about 8 months to about 11 months, about 8 months to about 10 months, about 8 months to about 9 months, about 9 months to about 12 months, about 9 months to about 11 months, about 9 months about 10 months, about 10 months to about 12 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, one or more doses are administered by subcutaneous administration. In some embodiments of any of the methods described herein, one or more doses are administered by intramuscular administration.

In some embodiments of any of the methods described herein, the two or more doses are administered from about 1 year to about 60 years (eg, from about 1 year to about 55 years, from 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 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 ~ 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 10 years to about 20 years, about 10 years to about 15 years, about 15 years to about 60 years , about 15 to about 55 years, about 15 to about 50 years, about 15 to about 45 years, about 15 to about 40 years, about 15 to about 35 years, about 15 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 ~ 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 40 years, about 25 years to about 35 years, about 25 years to about 30 years, about 30 years to about 60 years, about 30 years to about 55 years , about 30 to about 50 years, about 30 to about 45 years, about 30 to about 40 years, about 30 to about 35 years, about 35 to about 60 years, about 35 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 ~ 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 ~ 60 years).

In some embodiments of these methods, each of the one or more doses is about 0.01 mg/kg to about 0.01 mg/kg of each agent that results in decreased activation of the TGF-beta receptor. about 10 mg/kg of each agent that results in a decrease in activation of the TGF-β receptor (e.g., about 0.01 mg/kg to about 9 mg/kg, about 0.01 mg/kg of each agent that results in a decrease in TGF-β receptor activation). to about 8 mg/kg, about 0.01 mg/kg to about 7 mg/kg, about 0.01 mg/kg to about 6 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 4 mg/kg, about 0.01 mg/kg to about 3 mg/kg, about 0.01 mg/kg to about 2 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 0.4 mg/kg. 5 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 9 mg/kg, from about 0.05 mg/kg to about 8 mg/kg, from about 0.05 mg/kg to about 7 mg/kg, from about 0.05 mg/kg to about 6 mg/kg, from about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 4 mg/kg, about 0.05 mg/kg to about 3 mg/kg, about 0.05 mg/kg to about 2 mg/kg, about 0.05 mg/kg to about 1 mg/kg kg, about 0.05 mg/kg to about 0.5 mg/kg, about 0.05 mg/kg to about 0.1 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 9 mg/kg, about 0.1 mg/kg to about 8 mg/kg, about 0.1 mg/kg to about 7 mg/kg, about 0.1 mg/kg to about 6 mg/kg, about 0.1 mg/kg to about 5 mg/kg kg, from about 0.1 mg/kg to about 4 mg/kg, from about 0.1 mg/kg to about 3 mg/kg, from about 0.1 mg/kg to about 2 mg/kg, from about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 0.5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 9 mg/kg, about 0.5 mg/kg to about 8 mg/kg, about 0.5 mg/kg to about 7 mg/kg, about 0.5 mg/kg to about 6 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 4 mg/kg, about 0 .5 mg/kg to about 3 mg/kg, about 0.5 mg/kg to about 2 mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 9 mg/kg, about 1 mg/kg to about 8 mg/kg, about 1 mg/kg to about 7 mg/kg, about 1 mg/kg to about 6 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 10 mg/kg, about 2 mg/kg to about 9 mg/kg, about 2 mg/kg to about 8 mg/kg, about 2 mg/kg to about 7 mg/kg, about 2 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3 mg/kg to about 9 mg/kg, about 3 mg/kg to about 8 mg/kg, about 3 mg/kg to about 7 mg/kg, about 3 mg/kg to about 6 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4 mg/kg to about 9 mg/kg, about 4 mg/kg to about 8 mg/kg, about 4 mg/kg to about 7 mg/kg, about 4 mg/kg to about 6 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 9 mg/kg, about 5 mg/kg to about 8 mg/kg, about 5 mg/kg to about 7 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6 mg/kg to about 9 mg/kg, about 6 mg/kg to about 8 mg/kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7 mg/kg to about 9 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8 mg/kg to about 9 mg/kg, or about 8 mg/kg to about 10 mg/kg).

In some embodiments of these methods, the single or initial administration of one or more agents that result in decreased TGF-β receptor activation is administered to a subject at least 30 years of age (e.g., at least 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 70, 75, or 80 years old).

In some embodiments of any of the methods described herein, the subject has an age-related disease (e.g., an age-related disease or condition described herein or known in the art). any of the above) or an inflammatory disease (eg, any of the age-related diseases or conditions described herein or known in the art). In some embodiments of any of the methods described herein, the subject is administered a chemotherapeutic agent (e.g., any of the chemotherapeutic agents described herein or known in the art) ) has never been treated before. In some embodiments of any of the methods described herein, the subject is a therapeutic agent that induces cellular senescence (e.g., among the additional therapeutic agents that induce cellular senescence described herein) have not been previously treated for

Some embodiments of the methods described herein use one or more common gamma chain family cytokine receptor activators one or more times (e.g., three or more, four or more, five or more) , 6 or more, 7 or more, 8 or more, 9 or more, 10 or more) doses to the subject. In some embodiments of these methods, any two sequential doses of the two or more doses are spaced about one week to about one year apart (eg, about one week to about 11 months, about one week ~ about 10 months, about 1 week to about 9 months, about 1 week to about 8 months, about 1 week to about 7 months, about 1 week to about 6 months, about 1 week to about 5 months, about 1 week to about 4 months, about 1 week to about 3 months, about 1 week to about 2 months, about 1 week to about 1 month, about 1 week to about 3 weeks, about 1 week to about 2 weeks, about 2 weeks to about 12 months , about 2 weeks to about 11 months, about 2 weeks to about 10 months, about 2 weeks to about 9 months, about 2 weeks to about 8 months, about 2 weeks to about 7 months, about 2 weeks to about 6 months, about 2 weeks to about 5 months, about 2 weeks to about 4 months, about 2 weeks to about 3 months, about 2 weeks to about 2 months, about 2 weeks to about 1 month, about 2 weeks to about 3 weeks, about 3 weeks ~ about 12 months, about 3 weeks to about 11 months, about 3 weeks to about 10 months, about 3 weeks to about 9 months, about 3 weeks to about 8 months, about 3 weeks to about 7 months, about 3 weeks to about 6 months, about 3 weeks to about 5 months, about 3 weeks to about 4 months, about 3 weeks to about 3 months, about 3 weeks to about 2 months, about 3 weeks to about 1 month, about 1 month to about 12 months , about 1 month to about 11 months, about 1 month to about 10 months, about 1 month to about 9 months, about 1 month to about 8 months, about 1 month to about 7 months, about 1 month to about 6 months, about 1 month to about 5 months, about 1 month to about 4 months, about 1 month to about 3 months, about 1 month to about 2 months, about 2 months to about 12 months, about 2 months to about 11 months, about 2 months ~ about 10 months, about 2 months to about 9 months, about 2 months to about 8 months, about 2 months to about 7 months, about 2 months to about 6 months, about 2 months to about 5 months, about 2 months to about 4 months, about 2 months to about 3 months, about 3 months to about 12 months, about 3 months to about 11 months, about 3 months to about 10 months, about 3 months to about 9 months, about 3 months to about 8 months , about 3 months to about 7 months, about 3 months to about 6 months, about 3 months to about 5 months, about 3 months to about 4 months, about 4 months to about 12 months, about 4 months to about 11 months, about 4 months to about 10 months, about 4 months to about 9 months, about 4 months to about 8 months, about 4 months to about 7 months, about 4 months to about 6 months, about 4 months to about 5 months, about 4 months ~ about 4 months, about 5 months to about 12 months, about 5 months to about 11 months, about 5 months to about 10 months, about 5 months to about 9 months, about 5 months to about 8 months, about 5 months to about 7 months, about 5 months to about 6 months, about 6 months to about 12 months, about 6 months to about 11 months, about 6 months to about 10 months, about 6 months to about 9 months, about 6 months to about 8 months , about 6 months to about 7 months, about 7 months to about 12 months, about 7 months to about 11 months, about 7 months to about 10 months, about 7 months to about 9 months, about 7 months to about 8 months, about 8 months to about 12 months, about 8 months to about 11 months, about 8 months to about 10 months, about 8 months to about 9 months, about 9 months to about 12 months, about 9 months to about 11 months, about 9 months about 10 months, about 10 months to about 12 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, one or more doses are administered by subcutaneous administration. In some embodiments of any of the methods described herein, one or more doses are administered by intramuscular administration.

In some embodiments of any of the methods described herein, the two or more doses are administered from about 1 year to about 60 years (eg, from about 1 year to about 55 years, from 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 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 ~ 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 10 years to about 20 years, about 10 years to about 15 years, about 15 years to about 60 years , about 15 to about 55 years, about 15 to about 50 years, about 15 to about 45 years, about 15 to about 40 years, about 15 to about 35 years, about 15 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 ~ 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 40 years, about 25 years to about 35 years, about 25 years to about 30 years, about 30 years to about 60 years, about 30 years to about 55 years , about 30 to about 50 years, about 30 to about 45 years, about 30 to about 40 years, about 30 to about 35 years, about 35 to about 60 years, about 35 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 ~ 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 ~ 60 years).

In some embodiments of these methods, each of the one or more doses is from about 0.01 mg/kg of each common gamma chain family cytokine receptor activator to each common gamma chain family cytokine receptor activity about 10 mg/kg of each common gamma chain family cytokine receptor activator, for example about 0.01 mg/kg to about 9 mg/kg, about 0.01 mg/kg to about 8 mg/kg, about 0.01 mg/kg to about 7 mg/kg, about 0.01 mg/kg to about 6 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 4 mg/kg, about 0.01 mg/kg to about 3 mg/kg, about 0.01 mg/kg to about 2 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 0.1 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 9 mg/kg, about 0.05 mg/kg to about 8 mg/kg, about 0.05 mg/kg to about 7 mg/kg, about 0.05 mg/kg to about 6 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 4 mg/kg, about 0.05 mg/kg to about 3 mg/kg, about 0.05 mg/kg to about 2 mg/kg, about 0.05 mg/kg to about 1 mg/kg, about 0.05 mg/kg to about 0.4 mg/kg. 5 mg/kg, about 0.05 mg/kg to about 0.1 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 9 mg/kg, about 0.1 mg/kg to about 8 mg/kg, about 0.1 mg/kg to about 7 mg/kg, about 0.1 mg/kg to about 6 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 4 mg/kg kg, about 0.1 mg/kg to about 3 mg/kg, about 0.1 mg/kg to about 2 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 0.5 mg/kg. kg, from about 0.5 mg/kg to about 10 mg/kg, from about 0.5 mg/kg to about 9 mg/kg, from about 0.5 mg/kg to about 8 mg/kg, from about 0.5 mg/kg to about 7 mg/kg, about 0.5 mg/kg to about 6 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 4 mg/kg, about 0.5 mg/kg to about 3 mg/kg, about 0 .5 mg/kg to about 2 mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 9 mg/kg, about 1 mg/kg to about 8 mg/kg. kg, about 1 mg/kg to about 7 mg/kg, about 1 mg/kg to about 6 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 4 mg/kg, about 1 mg/kg to about 3 mg/kg. kg, about 1 mg/kg to about 2 mg/kg, about 2 mg/kg to about 10 mg/kg, about 2 mg/kg to about 9 mg/kg, about 2 mg/kg to about 8 mg/kg, about 2 mg/kg to about 7 mg/kg. kg, about 2 mg/kg to about 6 mg/kg, about 2 mg/kg to about 5 mg/kg, about 2 mg/kg to about 4 mg/kg, about 2 mg/kg to about 3 mg/kg, about 3 mg/kg to about 10 mg/kg. kg, about 3 mg/kg to about 9 mg/kg, about 3 mg/kg to about 8 mg/kg, about 3 mg/kg to about 7 mg/kg, about 3 mg/kg to about 6 mg/kg, about 3 mg/kg to about 5 mg/kg. kg, about 3 mg/kg to about 4 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4 mg/kg to about 9 mg/kg, about 4 mg/kg to about 8 mg/kg, about 4 mg/kg to about 7 mg/kg. kg, about 4 mg/kg to about 6 mg/kg, about 4 mg/kg to about 5 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 9 mg/kg, about 5 mg/kg to about 8 mg/kg. kg, about 5 mg/kg to about 7 mg/kg, about 5 mg/kg to about 6 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6 mg/kg to about 9 mg/kg, about 6 mg/kg to about 8 mg/kg. kg, about 6 mg/kg to about 7 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7 mg/kg to about 9 mg/kg, about 7 mg/kg to about 8 mg/kg, about 8 mg/kg to about 10 mg/kg. kg, about 8 mg/kg to about 9 mg/kg, or about 8 mg/kg to about 10 mg/kg).

In some embodiments of these methods, a single or first dose of one or more common gamma chain family cytokine receptor activators is administered to a subject at least 30 years of age (e.g., at least 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 70, 75, or 80 years old).

In some embodiments of any of the methods described herein, the subject has an age-related disease (e.g., an age-related disease or condition described herein or known in the art). any of the above) or an inflammatory disease (eg, any of the age-related diseases or conditions described herein or known in the art). In some embodiments of any of the methods described herein, the subject is administered a chemotherapeutic agent (e.g., any of the chemotherapeutic agents described herein or known in the art) ) has never been treated before. In some embodiments of any of the methods described herein, the subject is a therapeutic agent that induces cellular senescence (e.g., among the additional therapeutic agents that induce cellular senescence described herein) have not been previously treated for

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Immune Stimulation in C57BL/6 Mice Using Multichain Polypeptides Materials and Methods The first polypeptide comprises two TGFβRII domains, a human tissue factor 219 fragment, and a soluble form of human IL-15. Exemplary multichain comprising a first polypeptide and a second polypeptide that are fusions and the second polypeptide is a soluble fusion of two TGFβRII domains and the sushi domain of the human IL-15Rα chain A polypeptide (type A multichain polypeptide as described herein) was produced.

Results Immune Stimulation in C57BL/6 Mice Wild-type C57BL/6 mice were treated with either control PBS solution or dosages of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg of the multichain polypeptide, respectively. It was treated subcutaneously with After 4 days of treatment, spleen weight and percentage of various immune cell types present in the spleen were assessed. Specifically, single splenocyte suspensions were generated and stained with fluorochrome-conjugated antibodies including anti-CD4, anti-CD8, anti-NK1.1, and anti-CD19. The percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, natural killer (NK) cells, and CD19 ⁺ B cells present in the spleens of control-treated mice and mice treated with either multichain polypeptide was analyzed by flow cytometry. was evaluated using As shown in FIG. 1A, spleen weight in mice treated with multichain polypeptide increased with increasing dosage of multichain polypeptide. Furthermore, spleen weights in mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of multichain polypeptide, respectively, were significantly higher compared to mice treated with control solution. As shown in FIG. 1B, in the spleens of mice treated with multichain polypeptides, the percentage of both CD8 ⁺ T cells and NK cells increased with increasing dosage of multichain polypeptides. Specifically, the percentage of CD8 ⁺ T cells was higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10 mg/kg of the multichain polypeptide compared to control-treated mice, and NK cells increased. Percentages were higher in mice treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg of multichain polypeptide compared to control treated mice. These results demonstrate that exemplary multichain polypeptides can stimulate immune cells in the spleen, particularly CD8 ⁺ T cells and NK cells.

Pharmacokinetics Pharmacokinetics of exemplary multichain polypeptides were evaluated in wild-type C57BL/6 mice. Mice were treated subcutaneously with a dosage of 3 mg/kg multichain polypeptide. Blood was drawn from the tail vein at various time points and serum was prepared. Concentrations of multichain polypeptides in serum were determined by ELISA. Briefly, multichain polypeptides were captured using anti-human tissue factor antibody and detected using biotinylated anti-human TGFβ receptor, peroxidase-conjugated streptavidin, and ABTS substrate. Results indicated that the half-life of the exemplary multi-chain polypeptide was 12.66 hours.

Immune Stimulation in C57BL/6 Mice Over Time To assess the effects of immune stimulation with multichain polypeptides over time, mice were treated with a single dose of 3 mg/kg multichain polypeptide and spleen weights were measured. and the percentage of immune cell types present in the spleen were assessed immediately after treatment and at 16, 24, 48, 72, and 92 hours after treatment using the techniques described above. As shown in Figure 2A, spleen weights of mice treated with multichain polypeptides increased at 48 hours after treatment and continued to increase over 44 hours. Furthermore, as shown in FIG. 2B, in the spleen weights of mice treated with multichain polypeptides, the percentages of both CD8 ⁺ T cells and NK cells increased at 48 hours after treatment and increased over a further 44 hours. kept doing. These results further demonstrate that exemplary multichain polypeptides can stimulate splenic immune cells, particularly CD8 ⁺ T cells and NK cells over time.

Increased Proliferation and Granzyme B Expression by CD8 ⁺ T Cells and NK Cells To assess potential immune cell proliferation and cytotoxicity induced by multichain polypeptides, mice were treated with 3 mg/kg of multichain polypeptides. Treated with a single dose of peptide, the spleens of these mice were assessed immediately after treatment and at 16, 24, 48, 72, and 92 hours after treatment. Briefly, single splenocyte suspensions were generated and fluorochrome-labeled antibodies to various cell types, including anti-CD4, anti-CD8, anti-NK1.1, and anti-CD19, as well as anti-Ki67 antibodies (i.e., cell proliferation marker) and anti-granzyme B antibody (ie, cytotoxicity marker). Mean fluorescence intensity (MFI) of Ki67 and granzyme B of each immune cell type was analyzed by flow cytometry. As shown in Figures 3A and 3B, Ki67 and granzyme B expression by NK cells showed an increase at each time point assessed at 24 hours and thereafter compared to immediately after treatment (0 hours). In addition, Ki67 and Granzyme B expression by CD8 ⁺ T cells showed an increase at each time point assessed at 48 hours and thereafter compared to immediately after treatment (0 hours). Thus, a single dose of multichain polypeptide resulted in proliferation of CD8 ⁺ T cells and NK cells for at least 4 days after treatment.

These results indicate that multichain polypeptides not only increase the number of CD8 ⁺ T cells and NK cells, but also enhance the cytotoxicity of these cells.

Cytotoxicity to Tumor Cells Cytotoxicity of splenocytes activated by multichain polypeptides to tumor cells was next evaluated in C57BL/6 mice. Mouse Moloney leukemia cells (Yac-1) were labeled with CellTrace Violet and used as tumor target cells. C57BL/6 mice were treated with a single dose of 3 mg/kg multichain polypeptide and splenocytes were subsequently prepared at various time points and used as effector cells. Targeted tumor cells were mixed with effector cells at an effector:target (E:T) ratio of 10:1 and incubated at 37°C for 20 hours. Target cell viability was assessed by analysis of propidium iodide (PI)-positive, violet-labeled Yac-1 cells using flow cytometry. The percentage of Yac-1 tumor inhibition was calculated using the following formula.
Percentage of Yac-1 tumor inhibition=(1−number of viable Yac-1 cells in experimental sample/number of viable Yac-1 cells in sample without splenocytes)×100

As shown in FIG. 4, splenocytes from mice after treatment with multichain polypeptides for more than 24 hours showed increased cytotoxicity to Yac-1 cells compared to splenocytes from untreated mice. rice field.

Example 2: Immune Stimulation in C57BL/6 Mice Using a High Fat Diet-Based Type 2 Diabetes Mouse Model Materials and Methods TGFRt15-TGFRs are soluble human TGFβRII dimers (aa24-159)2 A multi-chain chimeric polypeptide (type A multi-chain chimeric polypeptide described herein) comprising two TGFβ binding domains. 21t15-TGFR is a multi-chain chimeric polypeptide (type A multi-chain chimeric polypeptide described herein) comprising IL-21 and TGFβ binding domains. 2t2 is a chimeric polypeptide (type B chimeric polypeptide described herein) comprising two IL-2 polypeptides.

Results To evaluate the effects of TGFRt15-TGFRs, 2t2, and 21t15-TGFRs in treating type 2 diabetes, a high-fat diet-based type 2 diabetes mouse model (B6.129P2-ApoE ^tmlUnc /J from The Jackson Laboratory) was used. It was used. Mice were fed either a control diet or a high fat diet for 11 weeks. A subset of mice fed a high-fat diet were also treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs. Mice on a control diet, a high-fat diet, and mice on a high-fat diet and treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs were evaluated 4 days after treatment. Briefly, single splenocyte suspensions were generated and stained with fluorochrome-conjugated antibodies including anti-CD4, anti-CD8, anti-NK1.1, and anti-CD19. The percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, natural killer (NK) cells, and CD19 ⁺ B cells present in the spleen of mice in each group was assessed using flow cytometry.

As shown in FIG. 5A, the percentage of NK cells in PBMCs was significantly increased after treatment with TGFRt15-TGFRs or 2t2 in mice fed a high-fat diet compared to untreated mice, whereas 21t15-TGFRs did not increase after treatment with Furthermore, the percentage of CD8 ⁺ T cells in PBMCs was significantly increased after treatment with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs compared to untreated mice. In addition, proliferation of PBMC CD4 ⁺ T cells, CD8 ⁺ T cells, natural killer (NK) cells, and CD19 ⁺ B cells was also assessed using anti-Ki67 antibody. As shown in FIG. 5B, the numbers of proliferating NK cells, CD4 ⁺ T cells, and CD8 ⁺ T cells were significantly increased after treatment with TGFRt15-TGFRs, but not after treatment with 2t2 or 21t15-TGFRs. did not increase.

To examine the effects of TGFRt15-TGFRs, 2t2, and 21t15-TGFRs on the appearance and texture of animal skin and hair, mice were fed a control or high-fat diet for 7 weeks, and a subset of the high-fat-fed mice was were also treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs. After one week of treatment, mice were evaluated for their appearance. High-fat diet-fed untreated mice, or high-fat diet-fed mice treated with 21t15-TGFRs, appeared ungroomed and disheveled, compared to control-fed mice. As a result, gray hair/hair loss increased (FIGS. 6A, 6B, and 6E). Surprisingly, high-fat diet-fed mice that received TGFRt15-TGFRs or 2t2 treatment compared with high-fat diet-fed mice that did not receive TGFRt15-TGFRs or 2t2 treatment (Fig. 6B). , groomed and looked healthy (less gray/hair loss) (FIGS. 6C and 6D). Specifically, TGFRt15-TGFRs or 2t2 treated mice exhibited superior skin and hair appearance and texture compared to control mice. These results indicate that treatment with TGFRt15-TGFRs or 2t2 improves the appearance and texture of mammalian skin and hair.

Mice were then fed a control or high-fat diet for 9 weeks, and a subset of the high-fat diet-fed mice were treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs. Four days after treatment, fasting body weights of mice in each group were measured. Fasting body weights of high-fat-fed, untreated mice, as well as high-fat-fed, 21t15-TGFRs-treated mice were significantly increased compared to control-fed mice. However, the fasting body weight of mice fed a high-fat diet and treated with TGFRt15-TGFRs or 2t2 was decreased compared to the other two high-fat diet groups. Fasting body weights of mice at the end of the study (9 weeks) are shown in FIG.

To assess fasting glucose levels in each group of mice, mice were fed either a control diet or a high-fat diet and were untreated or TGFRt15-TGFRs, 2t2, or 21t15 on days 44, 59, and 73. - treated with TGFRs; Fasting blood glucose levels of mice in each group were measured after 4 days of treatment. As shown in FIG. 8, fasting blood glucose levels after the second and third doses (days 59 and 73, respectively) were elevated in mice fed a high-fat diet but untreated (yellow line). ) in mice fed a high-fat diet and treated with 2t2 (red line). Fasting blood glucose levels remained constant in mice fed a high-fat diet and treated with TGFRt15-TGFRs (green line), whereas fasting blood glucose levels remained constant in mice fed a high-fat diet and treated with 21t15-TGFRs (green line). was elevated in mice treated with (blue line).

Example 3 Chemotherapy-Induced Senescent B16F10 Melanoma Cells Express NK Ligands Materials and Methods Cellular senescence of B16F10 melanoma cells was investigated by treating cells with docetaxel (7.5 μM, Sigma) for 3 days. After treatment, they were induced by recovery in complete medium for 4 days. Cellular senescence was accessed by staining cells with senescence-associated β-galactosidase (SA β-gal). Briefly, B16F10 control and senescent cells (B16F10-SNC) were washed once with PBS and fixed with 0.5% glutaraldehyde (PBS (pH 7.2)) for 30 minutes. Cells were washed with X-gal solution (1 mg/mL X-gal, 0.12 mM K ₃ Fe[CN] ₆ , 0.12 mM K ₄ Fe[CN] ₆ and 1 mM MgCl ₂ (pH 6.0) in PBS. 0)) at 37° C. overnight and imaged using a Nikon light microscope.

Results Cellular senescence of B16F10 melanoma cells was induced using chemotherapy as described above. As shown in FIG. 9A, chemotherapy-induced senescent B16F10 cells (B16F10-SNC) were positive for SA β-gal staining, whereas control B16F10 cells did not stain. Expression of senescent genes was then analyzed using RT-qPCR using RNA isolated after induction of senescence on day 0 or 4, 8, 12, 16, respectively. Expression levels were normalized to control B16F10 cells. As shown in Figures 9B-9D, expression of p21, IL6, and DPP4 was upregulated in RNA isolated from senescent cells during the experimental period. Furthermore, as shown in FIGS. 9E and 9F, the expression of RATE1E and ULBP1 (an NK-activating receptor NKG2D ligand) was also induced in senescent cells, with the highest expression levels at day 16. These results indicate that chemotherapy-induced senescent B16F10 cells are exposed to greater cytotoxicity of activated NK cells than control B16F10 cells.

Acquisition of Stem Cell Properties in Chemotherapy-Induced Senescent B16F10 Melanoma Cells To investigate whether chemotherapy-induced senescent B16F10 melanoma cells acquired stem cell properties, a colony formation assay was performed. Briefly, 1000 cells/well were seeded in 6 well plates and medium was changed every 3 days. After 5 weeks in culture, senescent cells were able to form colonies, as shown in FIG. 10A (image taken at 100× magnification). To assess stem cell marker expression by colonies, RNA was isolated from colonies and Oct4 and Notch4 mRNA expression was determined by RT-qPCR. Compared to control B16F10 cells, chemotherapy-induced senescent B16F10 melanoma cells showed upregulation of cancer stem cell markers Oct4 and Notch4 (FIGS. 10B and 10C). In addition, cell surface expression of stem cell markers CD44, CD24 and CD133 was assessed by flow cytometry after staining with antibodies against CD44, CD24 and CD133. As shown in FIGS. 10D-10F, chemotherapy-induced senescent stem cells (B16F10-SNC-CSCs) had double-positive populations (CD44 ⁺ CD24 ⁺ , CD44 ⁺ CD133 ⁺ , and CD24 ⁺ CD133 ⁺ ).

Chemotherapy-induced senescence with stem cell properties (CIS) melanoma cells are more 'migratory' and 'invasive' than control B16F10 cells Chemotherapy-induced senescence with stem cell properties (CIS) The migration of melanoma cells (B16F10-SNC-CSC) was analyzed using a migration assay. Briefly, control B16F10 cells and B16F10-SNC-CSC cells were seeded in 6-well plates and wounded with a p20 pipette tip. Cell movement was imaged after 0, 12, and 24 hours. As shown in FIG. 11A, chemotherapy-induced senescence (CIS) melanoma cells with stem cell properties (B16F10-SNC-CSC) migrated more in an in vitro migration assay compared to control B16F10 cells.

Next, the invasion properties of chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSCs) were analyzed using an invasion assay. Invasion assays were performed in 24-well transwell inserts coated with Matrigel. Briefly, 0.5×10 ⁶ control B16F10 cells and B16F10-SNC-CSC cells were seeded in serum-free medium in the upper chamber and the lower chamber was filled with medium supplemented with 10% FBS. After 16 hours of incubation, the cells on the top surface of the filters were removed and the cells below the filters were fixed and stained with 0.02% crystal violet solution. Cell numbers were counted in three fields at 100x magnification. As shown in FIGS. 11B and 11C, chemotherapy-induced senescent cells with stem cell properties were more aggressive in invading Matrigel-coated membranes compared to control B16F10 cells. These results indicate that chemotherapy-induced senescent B16F10 tumor cells regained their proliferative capacity and acquired stem cell properties, increasing their migratory capacity and invasiveness to metastasis.

Cytotoxic activity of mouse NK cells against chemotherapy-induced senescent cells with stem cell properties To expand NK cells in vivo, C57BL/6 mice were injected subcutaneously with TGFRt15-TGFRs (10 mg/kg) for 4 days. Spleens were harvested from these mice and NK cells were purified using MACS Miltenyi columns. Purified NK cells were then expanded in vitro with 2t2 (Fig. 12A).

To assess the cytotoxicity of expanded NK cells, chemotherapy-induced senescent stem cells (B16F10-SNC-CSC) or control B16F10 cells were labeled with CellTrace violet and activated 2t2 mouse NK cells in vitro. (isolated from spleens of C57BL/6 injected with 10 mg/kg TGFRt15-TGFRs for 4 days) and incubated at various E:T ratios for 16 hours. B16F10-SNC-CSC and control B16F10 cells were trypsinized, washed, resuspended in complete medium containing propidium iodide (PI) solution and accessed by cytotoxicity by flow cytometry. As shown in FIG. 12B, NK cells were more effective in killing chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) compared to control B16F10 cells.

Combination Therapy in a Mouse Model of Melanoma The effect of TGFRt15-TGFRs in treating melanoma was evaluated in a mouse model of melanoma. Briefly, 5×10 ⁵ B16F10 cells were injected subcutaneously into C57BL/6 mice. When tumor volumes reached approximately 100 mm ³ , mice were treated with docetaxel (chemotherapy) (5 mg/kg) or TA99 (200 μg) alone or in combination every 3 days, and TGFRt15-TGFRs (3 mg/kg) weekly. One application was applied (Fig. 13A). Mice receiving saline, docetaxel (chemotherapy)/TA99 alone, or TGFRt15-TGFRs alone were used as controls. Five mice were tested in each experimental and control group. Tumor volumes were measured every 3 days. As shown in FIGS. 13B and 13C, the combination of TGFRt15-TGFRs and chemotherapy or TA99 was significantly more effective in a syngeneic melanoma mouse model compared to mice treated with saline or chemotherapy or TA99 alone. slowed tumor progression.

Example 4: Chemotherapeutic induction of senescence in the human pancreatic cell line SW1990 Materials and methods β-galactosidase staining: Confirmation of chemotherapy-induced senescence was obtained using a commercially available kit (Cell Signaling Technology) from the manufacturer. and standard β-galactosidase staining was performed at pH 6.0. The next day, staining solution was removed, cells were washed with phosphate buffered saline, and 70% glycerol was added to the wells. β-galactosidase positive cells stain blue, while control untreated cells do not.

Flow cytometry: One million control and senescent cells were obtained and stained using commercially available antibodies against stem cell surface markers such as anti-CD44 and anti-CD24 antibodies (Biolegend) according to the manufacturer's instructions. Cells were then washed and analyzed using a BD Celesta flow cytometer. Cells exhibiting stem cell-like properties become doubly positive for both CD44 and CD24.

Gene Expression Assays: One million control and senescent cells were obtained and lysed using Trizol (Thermofisher) followed by RNA purification using an RNA isolation kit (Qiagen). RNA was quantified and converted to cDNA using the Qiagen cDNA Quantitect kit. The cDNA was then used as a template for standard Taqman gene expression assays (Thermofisher) to quantify the relative abundance of senescent stem cell markers and NK ligands.

NK cell cytotoxicity assay: NK cells were isolated from healthy human donors (n=2) using a commercial NK isolation kit (stem cells) and treated overnight with cytokine fusion molecule 18t15-12s (100 nM). activated. The following day, NK cells were washed to remove cytokine molecules and mixed with either CellTrace Violet-labeled control untreated tumor cells or chemotherapy-induced senescent tumor cells at an E:T ratio of 4:1 for 20 hours. The next day, cells were trypsinized and the complete contents of each well were analyzed using flow cytometry to analyze percent inhibition of cells.

Results Senescence was induced in the human pancreatic tumor cell line SW1990 by treatment with the chemotherapeutic drugs Abraxane (Celgene) and gemcitabine (Sigma Aldrich) for 3 days at 2.5 μM and 6.25 μM, respectively. Untreated SW1990 cells were used as a control. The medium was changed after 3 days and the cells were left in culture medium for 4 days. As shown in Figure 14, senescent cells treated with chemotherapy drugs were positive for β-galactosidase staining (blue), whereas control cells did not stain. Senescent and control cells were evaluated for expression of senescence and stem cell markers at 4, 11, and 22 days after treatment. As shown in Figure 14, senescent cells showed an increase in double-positive CD44 and CD24 staining over time compared to control cells. In addition, chemotherapy-induced senescent SW1990 cells were 0 after treatment using senescence markers including DPP4, IL6, and p21, stem cell markers including Oct 3/4, CD24, and CD44, and the gene expression assays described above. The expression of NK ligands including Nectin and MICA on days 2, 4 and 24 was also analyzed. As shown in Figure 15, the expression of all mentioned markers showed an increase over time.

Cytotoxicity of In Vitro Activated Human NK Cells To assess the cytotoxicity of in vitro activated human NK cells (treated with 18t15-12s), the chemotherapeutic drugs Abraxane (Celgene) and gemcitabine (Sigma) Aldrich) induced senescence in the human pancreatic tumor cell line SW1990 by treatment with 2.5 μM and 6.25 μM for 3 days, respectively. Untreated SW1990 cells were used as a control. The medium was changed after 3 days and the cells were left in culture medium for 30 days. Culture medium was changed every 4 days. Activated NK cells were obtained and their cytotoxicity against chemotherapy-induced senescent tumor cells and untreated control tumor cells was assessed using the NK cell cytotoxicity assay described above. As shown in Figure 16, activated NK cells showed increased cytotoxicity against both control SW1990 cells (SW1990) and senescent SW1990 cells (SW1990s).

Example 5 Generation of IL-12/IL-15RαSu DNA Construct In a non-limiting example, an IL-12/IL-15RαSu DNA construct was generated (FIG. 17). Human IL-12 subunit sequence, human IL-15RαSu sequence, human IL-15 sequence, human tissue factor 219 sequence, and human IL-18 sequence were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. . A GS(3) linker was used to join IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) to generate a single-chain version of IL-12, followed by the IL-12 sequence. A DNA construct was made by directly linking to the IL-15RαSu sequence. The final IL-12/IL-15RαSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence (including signal peptide sequence) of the IL12/IL-15RαSu construct is as follows (SEQ ID NO: 181).
(signal peptide)
ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC
(Human IL-12 subunit beta (p40))
ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGAACCCTCACAATCCAAGTTAA GGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGGAGGCGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAAAACTACAGCGGG TCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAGCAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGCGATACAAGGAATACGAGTACAGCGTGGAGTGCCAAGA AGATAGCGCTTGTCCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAAACTCAAGTACGAGAACTACACCCTCCTCCTTCTTATCCGGGACATCATTAAGCCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAATAGCCGGCAAGTTGAGGTCT CTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCTCCATCAGCGTGAGGGCTCAAGATC GTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC
(linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(Human IL-12 subunit alpha (p35))
CGTAACCTCCCCGTGGCTACCCCCGATCCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAAGATGTACCAAGTGGAGTTCAAGACCCATGAACGCCAA GCTGCTCATGGACCCTAAACGGCAGATCTTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTACTCCACGCCTTTAGGATCCG GGCCGTGACCATTGACCGGGTCATGAGCCTATTTAAAACGCCAGC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

Example 6: Generation of an IL-18/TF/IL-15 DNA Construct In a non-limiting example, an IL-18 sequence was ligated to the N-terminal coding region of tissue factor 219 and the IL-18/TF construct was converted to IL-18. An IL-18/TF/IL-15 construct was generated by joining the N-terminal coding regions of 15 (FIG. 18). The nucleic acid sequence (including leader sequence) of the IL-18/TF/IL-15 construct synthesized by Genewiz is as follows (SEQ ID NO: 177).
(signal peptide)
ATGAAGTGGGTCACATTTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC
(human IL-18)
TACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCCGGGGCAT GGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTAC GAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTTCCATCATGTTCACCGTCCAAAACGAGGAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

Example 7: Secretion of IL-12/IL-15RαSu and IL-18/TF/IL-15 fusion proteins. -1 modified retroviral expression vector (as described in Hughes, Hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells resulted in the formation of a soluble IL-18/TF/IL-15:IL-12/IL-15RαSu protein complex (designated 18t15-12s, FIGS. 19 and 20). formation and secretion of The 18t15-12s protein was purified from CHO-K1 cell culture supernatants using anti-TF antibody affinity chromatography and size exclusion chromatography to isolate IL-12/IL-15RαSu fusion protein and IL-18/TF/IL-18t15-12s protein. A soluble (non-aggregated) protein complex consisting of 15 fusion proteins was obtained.

The amino acid sequence (including signal peptide sequence) of the IL12/IL-15RαSu fusion protein is as follows (SEQ ID NO: 180).
(signal peptide)
MKWVT FISLLFLFS SAYS
(Human IL-12 subunit beta (p40))
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCS
(linker)
GGGGSGGGGSGGGGGS
(Human IL-12 subunit alpha (p35))
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSSIYEDLKMYQVEFKTMNAKLMDPKRQIFLDQNMLAAVIDELMQALN FNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence (including signal peptide sequence) of the IL-18/TF/IL-15 fusion protein is as follows (SEQ ID NO: 176).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-18)
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILLKEDELGDRSIMFT VQNED
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

Example 8: Purification of 18t15-12s by immunoaffinity chromatography An anti-TF antibody affinity column was connected to a GE Healthcare™ AKTA Avant protein purification system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

The 18t15-12s cell culture harvest was adjusted to pH 7.4 with 1 M Tris base and loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). Absorbance at 280 nm was collected, after which samples were neutralized to pH 7.5-8.0 by addition of 1 M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon® centrifugal filters with a molecular weight cutoff of 30 KDa. Figure 21 shows that the 18t15-12s conjugate binds to an anti-TF antibody affinity column, where TF is the 18t15-12s binding partner. Buffer-exchanged protein samples are stored at 2-8° C. for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 10 column volumes of PBS, 0.05% sodium azide and stored at 2-8°C.

Example 9: Size Exclusion Chromatography of 18t15-12s A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant Protein Purification System. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 200 μL of 1 mg/mL 18t15-12s complex onto the column. Injections were followed with 1.25 column volumes of PBS. SEC chromatograph is shown in FIG. There is a major 18t15-12s protein peak along with a minor high molecular weight peak, probably due to different degrees of glycosylation of 18t15-12s dimers or aggregates.

Example 10: SDS-PAGE of 18t15-12s
Purified 18t15-12s protein samples were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE to determine purity and protein molecular weight. Gels were stained with InstantBlue™ for approximately 30 minutes and then destained overnight in purified water. FIG. 23 shows an example of an SDS gel of anti-TF antibody affinity purified 18t15-12s with bands at the expected molecular weights (66 kDa and 56 kDa).

Example 11: Glycosylation of 18t15-12s in CHO-K1 Cells Glycosylation of 18t15-12s in CHO-K1 cells was confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs) according to the manufacturer's instructions. FIG. 24 shows examples of SDS PAGE of deglycosylated and non-deglycosylated 18t15-12s. As seen in lane 4 of FIG. 24, deglycosylation reduces the molecular weight of 18t15-12s.

Example 12: Recombinant Protein Quantification of 18t15-12s Complexes 18t15-12s complexes were detected and quantified using standard sandwich ELISA methods (Figures 25-28). An anti-human tissue factor antibody served as the capture antibody and a biotinylated anti-human IL-12, IL-15, or IL-18 antibody (BAF219, BAM247, D045-6, all R&D Systems) served as the detection antibody. Tissue factor in the purified 18t15-12s protein complex was also detected using anti-human tissue factor capture antibody (I43) and anti-human tissue factor antibody detection. The I43/anti-TF antibody ELISA was compared to similar concentrations of purified tissue factor.

Example 13: Immunostimulatory Potential of 18t15-12s Complexes To assess the IL-15 immunostimulatory activity of 18t15-12s complexes, increasing concentrations of 18t15-12s were added to 200 μL of IMDM:32Dβ cells in 10% FBS medium. (104 cells/well). 32Dβ cells were incubated at 37° C. for 3 days. On day 4, WST-1 proliferation reagent (10 μL/well) was added and after 4 hours absorbance was measured at 450 nm to measure cell proliferation based on the 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 Figure 29, 18t15-12s exhibited IL-15 dependent cell proliferation of 32Dβ cells. The 18t15-12s complex exhibited reduced activity compared to human recombinant IL-15, possibly due to binding 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 mixed with HEK-Blue IL-12 and HEK in 200 μL of IMDM:10% heat-inactivated FBS medium. - added to Blue IL-18 reporter cells (5 x ¹⁰⁴ cells/well, hkb-il12 and hkb-hmil18, InvivoGen). Cells were incubated overnight at 37°C. 20 μl of induced HEK-Blue IL-12 and HEK-Blue IL-18 reporter cell supernatant was added to 180 μl of QUANTI-Blue (InvivoGen) and incubated at 37° C. for 1-3 hours. IL-12 or IL-18 activity was assessed by measuring absorbance at 620 nm. Human recombinant IL-12 or IL-18 were evaluated as positive or negative controls. As shown in Figures 30 and 31, the cytokine domains of the 18t15-12s complex each retain specific biological activities. The activity of 18t15-12s is likely due to binding of IL-15 and tissue factor to the IL-18 domain, and binding of IL-12 to the IL-15Rα sushi domain, resulting in increased binding of human recombinant IL-18 or IL-12. decreased compared to activity.

Example 14: Induction of Cytokine-Inducible Memory-Like NK Cells by 18t15-12s Complex mL), and can be induced ex vivo after overnight stimulation with IL-18 (50 ng/mL). These memory-like properties have been measured by increased expression of IL-2 receptor alpha (IL-2Rα, CD25), CD69 (and other activation markers), and IFN-γ production. To assess the ability of the 18t15-12s complex to promote the generation of cytokine-induced memory-like NK cells, purified human NK cells (>95% CD56+) were treated with 0.01 nM to 10000 nM of the 18t15-12s complex or Individual cytokine combinations (recombinant IL-12 (10 ng/ml), IL-18 (50 ng/ml), and IL-15 (50 ng/ml)) were stimulated 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 a blood bank and CD56+ NK cells were isolated using RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone ) resuspended at 0.2×10 ⁶ /mL in 96-well flat-bottom plates in medium (supplemented). Cells were treated with a mixture of cytokines hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D Systems), and hIL-15 (50 ng/mL) (NCI), or 0.01 nM-10000 nM. 18t15-12s at 37° C., 5% CO ₂ for 14-18 hours. Cells were then harvested and surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend) for 30 minutes. After staining, cells were washed in FACS buffer (containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). After two washes, cells were analyzed using a BD FACSCelesta™ flow cytometer (plotted data-mean fluorescence intensity, Figures 32A and 32B).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated using RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone ) resuspended at 0.2×10 ⁶ /mL in 96-well flat-bottom plates in medium (supplemented). Cells were treated with a cytokine mixture of hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D), and hIL-15 (50 ng/mL) (NCI), or 0.01 nM-10000 nM Either 18t15-12s complex was stimulated at 37° C., 5% CO ₂ for 14-18 hours. Cells were then treated with 10 μg/mL Brefeldin A (Sigma) and 1X monensin (eBioscience) for 4 hours, harvested and then 30-fold treated with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies. Stained for minutes. After staining, cells were washed in FACS buffer (containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes) and fixed at room temperature for 10 minutes. After fixation, cells were washed in 1× permeabilization buffer (eBioscience) (1500 RPM, 5 minutes at room temperature) and stained with IFN-γ-PE (Biolegend) for 30 minutes at room temperature. Cells were washed again with 1x permeabilization buffer and then with FACS buffer. Cell pellets were resuspended in 300 μl of FACS buffer and analyzed using a BD FACSCelesta™ flow cytometer (plot % IFN-γ positive cells, FIG. 33).

Example 15: In vitro cytotoxicity of NK cells against human tumor cells Human myeloid leukemia cells K562 (labeled with CellTrace violet) were treated with purified human Incubated with NK cells. After 20 hours, cultures were harvested, stained with propidium iodide (PI) and evaluated by flow cytometry. As shown in Figure 34, the 18t15-12s complex induced human NK cytotoxicity against K562 at similar or higher levels than the cytokine mixture, and both the 18t15-12s complex and the cytokine mixture It induced higher cytotoxicity than the medium control.

Example 16: Generation of IL-12/IL-15RαSu/αCD16scFv DNA Constructs and IL-18/TF/IL-15 DNA Constructs In a non-limiting example, IL-12/IL-15RαSu/αCD16scFv DNA constructs and IL-15RαSu/αCD16scFv DNA constructs A 18/TF/IL-15 DNA construct was generated (Figures 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 Genewiz. A GS(3) linker was used to join IL-12 subunit beta (p40) to IL-12 subunit alpha (p35) to generate a single-chain version of IL-12, translating the IL-12 sequence into IL- A DNA construct was made by directly joining the 15RαSu sequence and 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 (p40))
ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGAACCCTCACAATCCAAGTTAA GGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGGAGGCGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAAAACTACAGCGGG TCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAGCAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGCGATACAAGGAATACGAGTACAGCGTGGAGTGCCAAGA AGATAGCGCTTGTCCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAAACTCAAGTACGAGAACTACACCCTCCTCCTTCTTATCCGGGACATCATTAAGCCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAATAGCCGGCAAGTTGAGGTCT CTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCTCCATCAGCGTGAGGGCTCAAGATC GTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC
(linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(Human IL-12 subunit alpha (p35))
CGTAACCTCCCCGTGGCTACCCCCGATCCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAAGATGTACCAAGTGGAGTTCAAGACCCATGAACGCCAA GCTGCTCATGGACCCTAAACGGCAGATCTTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTACTCCACGCCTTTAGGATCCG GGCCGTGACCATTGACCGGGTCATGAGCCTATTTAAAACGCCAGC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
(anti-human CD16 light chain variable domain)
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG TTCTCCGGATCCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGCGCGGCACCAAGCTGACCGTGGGCCAT
(linker)
GGCGGCGGCGGCTCCGGAGGCGCGCGGCAGCGGCGGAGGAGGATCC
(anti-human CD16 heavy chain variable domain)
GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCCTCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGGTGAGGCAGGCTCCTGGAAAGGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGG CGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCG TGTCCAGG

Constructs were also made by joining the IL-18 sequence to the N-terminal coding region of tissue factor 219 and the IL-18/TF construct to the N-terminal coding region of IL-15 (Figure 36). The nucleic acid sequence (including leader sequence) of the IL-18/TF/IL-15 construct is as follows (SEQ ID NO: 177).
(signal peptide)
ATGAAGTGGGTCACATTTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC
(human IL-18)
TACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCCGGGGCAT GGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTAC GAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTTCCATCATGTTCACCGTCCAAAACGAGGAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

Example 17: Secretion of IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 Fusion Proteins IL-12/IL-15RαSu/αCD16scFv and IL-18/TF/IL-15 Constructs was cloned into a pMSGV-1 modified retroviral expression vector (Hughes, Hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells yielded a soluble IL-18/TF/IL-15:IL-12/IL-15RαSu/αCD16scFv protein complex (designated 18t15-12s/αCD16, FIG. 37 and Figure 38) resulted in secretion. Co-expression of these two constructs in CHO-K1 cells yielded a soluble IL-18/TF/IL-15:IL-12/IL-15RαSu/αCD16scFv protein complex (designated 18t15-12s/αCD16, FIG. 37 and Figure 38), which can be purified by anti-TF antibody 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 signal peptide sequence) of the IL-12/IL-15RαSu/αCD16scFv fusion protein is as follows (SEQ ID NO:225).
(signal peptide)
MKWVT FISLLFLFS SAYS
(Human IL-12 subunit beta (p40))
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCS
(linker)
GGGGSGGGGSGGGGGS
(Human IL-12 subunit alpha (p35))
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSSIYEDLKMYQVEFKTMNAKLMDPKRQIFLDQNMLAAVIDELMQALN FNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(anti-human CD16 light chain variable domain)
SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH
(linker)
GGGGSGGGGSGGGGGS
(anti-human CD16 heavy chain variable domain)
EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR

The amino acid sequence (including leader sequence) of the IL-18/TF/IL-15 fusion protein is as follows (SEQ ID NO:221).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-18)
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILLKEDELGDRSIMFT VQNED
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 18: Generation of IL-18/IL-15RαSu DNA Constructs and IL-12/TF/IL-15 DNA Constructs In a non-limiting example, IL-18 /IL-15RαSu DNA constructs and IL-12/TF/ An IL-15 DNA construct was made. 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 Genewiz. A DNA construct was made by directly conjugating IL-18 to IL-15RαSu. Additional constructs were also made by joining the IL-12 sequence to the N-terminal coding region of the human tissue factor 219 form and the IL-12/TF construct to the N-terminal coding region of IL-15. A single chain version of IL-12 (p40-linker-p35) was used as described above.

The nucleic acid sequence (including signal peptide sequence) of the IL-18/IL-15RαSu construct is as follows (SEQ ID NO:320).
(signal peptide)
ATGAAGTGGGTCACATTTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC
(human IL-18)
TACTTCGGCAAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCCGGGGCAT GGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACAAGATCATCTCTTTAAGGAAATGAACCCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTAC GAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTTCCATCATGTTCACCGTCCAAAACGAGGAT
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

The nucleic acid sequence (including leader sequence) of the IL-12/TF/IL-15 construct is as follows (SEQ ID NO:321).
(signal peptide)
ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC
(Human IL-12 subunit beta (p40))
ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGAACCCTCACAATCCAAGTTAA GGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGGAGGCGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAAAACTACAGCGGG TCGTTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTTCTCCGTGAAAGCAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGCGATACAAGGAATACGAGTACAGCGTGGAGTGCCAAGA AGATAGCGCTTGTCCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAAACTCAAGTACGAGAACTACACCCTCCTCCTTCTTATCCGGGACATCATTAAGCCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAAATAGCCGGCAAGTTGAGGTCT CTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCTCCATCAGCGTGAGGGCTCAAGATC GTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC
(linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(Human IL-12 subunit alpha (p35))
CGTAACCTCCCCGTGGCTACCCCCGATCCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGG CTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAAGATGTACCAAGTGGAGTTCAAGACCCATGAACGCCAA GCTGCTCATGGACCCTAAACGGCAGATCTTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTACTCCACGCCTTTAGGATCCG GGCCGTGACCATTGACCGGGTCATGAGCCTATTTAAAACGCCAGC
(human tissue factor 219)
AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTTGAGCAAGTTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

Example 19: Secretion of IL-18/IL-15RαSu and IL-12/TF/IL-15 fusion proteins Cloned into a modified retroviral expression vector (Hughes, Hum Gene Ther 16:457-72, 2005, incorporated herein by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells resulted in the secretion of a soluble IL-12/TF/IL-15:IL-18/IL-15RαSu protein complex (termed 12t15/s18). , which can be purified by anti-TF antibody affinity and other chromatographic methods.

The amino acid sequence (including signal peptide sequence) of the IL-18/IL-15RαSu fusion protein is as follows (SEQ ID NO:322).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-18)
YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILLKEDELGDRSIMFT VQNED
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence (including leader sequence) of the IL-12/TF/IL-15 fusion protein is as follows (SEQ ID NO:323).
(signal peptide)
MKWVT FISLLFLFS SAYS
(Human IL-12 subunit beta (p40))
IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRA QDRYYSSSWSEWASVPCS
(linker)
GGGGSGGGGSGGGGGS
(Human IL-12 subunit alpha (p35))
RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSSIYEDLKMYQVEFKTMNAKLMDPKRQIFLDQNMLAAVIDELMQALN FNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

Example 20: Recombinant Protein Determination of 18t15-12s16 Complexes 18t15-12s16 complexes (including IL-12/IL-15RαSu/αCD16scFv; IL-18/TF/IL-15) were assayed using standard sandwich ELISA methods. was detected and quantified using a (Figure 39). Anti-human tissue factor antibody/IL-2 or anti-TF antibody/IL-18 acted as a capture antibody, and biotinylated anti-human IL-12 or IL-18 antibody (BAF219, D045-6, both from R&D Systems) was detected. functioned as an antibody. Tissue factor was detected using anti-human tissue factor antibody (I43) and anti-human tissue factor antibody detection.

Example 21 Generation of TGFβRII/IL-15RαSu DNA Constructs and IL-21/TF/IL-15 DNA Constructs In a non-limiting example, a TGFβRII/IL-15RαSu DNA construct was generated (FIG. 40). Human TGFβRII dimer sequences and human IL-21 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. DNA constructs by joining TGFβRII to another TGFβRII using a linker to generate a single-chain version of TGFβRII and then directly joining the TGFβRII single-chain dimer sequence to the N-terminal coding region of IL-15RαSu. was made.

The nucleic acid sequence (including signal sequence) of the TGFβRII/IL-15RαSu construct is as follows (SEQ ID NO: 196).
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
((human TGFβRII-first fragment)
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT
(linker)
GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT
(Human TGFβRII-Second Fragment)
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC GACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC
(Human IL-15Rα sushi domain)
ATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTTGTACTCCAGGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAAGCCGGCACGTCCAGCCCTGACGGAGTGCGTGTTTGAACAAGGCCACGAATGTCGCCCCACT GGACAACCCCCCAGTCTCAAATGTATTAGA

In addition, IL-21/TF/IL-15 by linking the IL-21 sequence to the N-terminal coding region of tissue factor 219 and the IL-21/TF construct to the N-terminal coding region of IL-15. A construct was made (Figure 41). The nucleic acid sequence (including leader sequence) of the IL-21/TF/IL-15 construct is as follows (SEQ ID NO: 192).
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC
(human tissue factor 219)
TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAA ATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTTCTACGAGAATTCCCCCGAATTCACCCTTATTTAGAGACCAATTTAGGCCAGCCCTACCATCCAGAGCTTCGAGCCAAGTTGGCACCAAGG TGAACGTCACCGTCGAGGATGAAAAGGACTTTAGTGCGGCCGGAATAACACATTTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAA AGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

Example 22: Secretion of TGFβRII/IL-15RαSu and IL-21/TF/IL-15 fusion proteins Cloned into a viral expression vector (as described in Hughes et al., Hum Gene Ther 16:457-72, 2005, incorporated herein by reference), and the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells resulted in secretion of soluble IL-21/TF/IL-15:TGFβRII/IL-15RαSu protein complexes (termed 21t15-TGFRs, FIGS. 42 and 43). was brought. 21t15-TGFRs complexes were purified from CHO-K1 cell culture supernatants using anti-TF antibody affinity chromatography and other chromatographic methods.

The amino acid sequence (including signal peptide sequence) of the TGFβRII/IL-15RαSu construct is as follows (SEQ ID NO: 195).
(signal peptide)
MKWVT FISLLFLFS SAYS
(Human TGFβRII-first fragment)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPD
(linker)
GGGGSGGGGSGGGGGS
(Human TGFβRII-Second Fragment)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPD
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence (including signal peptide sequence) of the mature IL-21/TF/IL-15 fusion protein is as follows (SEQ ID NO: 191).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

Example 23: Purification of 21t15-TGFRs by immunoaffinity chromatography An anti-TF antibody affinity column was attached to a GE Healthcare AKTA™ Avant protein purification system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

A cell culture harvest of 21t15-TGFRs was adjusted to pH 7.4 with 1 M Tris base and loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). Absorbance at 280 nm was collected, after which samples were neutralized to pH 7.5-8.0 by addition of 1 M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon® centrifugal filters with a molecular weight cutoff of 30 KDa. Figure 44 shows that the 21t15-TGFRs conjugate binds to an anti-TF antibody affinity column, where TF is the 21t15-TGFRs binding partner. Buffer-exchanged protein samples are stored at 2-8° C. for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 10 column volumes of PBS, 0.05% sodium azide and stored at 2-8°C.

Example 24: Size Exclusion Chromatography of 21t15-TGFRs A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.8 mL/min. A capillary loop was used to inject 200 μL of 1 mg/mL 21t15-TGFRs conjugate onto the column. Injections were then followed with 1.25 column volumes of PBS. SEC chromatograph is shown in FIG. There were two protein peaks likely representing monomeric and dimeric forms of 21t15-TGFRs.

Example 25: SDS-PAGE of 21t15-TGFRs
To determine purity and protein molecular weight, purified 21t15-TGFRs complex protein samples were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE under reducing conditions. Gels were stained with InstantBlue™ for approximately 30 minutes and then destained overnight in purified water. FIG. 46 shows an example of an SDS gel of anti-TF antibody affinity purified 21t15-TGFRs with bands at 39.08 kDa and 53 kDa.

Glycosylation of 21t15-TGFRs in CHO cells was confirmed using the Protein Deglycosylation Mix II kit (New England Biolabs) and manufacturer's instructions. As seen in lane 4 of FIG. 46, deglycosylation reduces the molecular weight of 21t15-TGFRs.

Example 26: Recombinant Protein Quantification of 21t15-TGFRs Complexes 21t15-TGFRs complexes were detected and quantified using standard sandwich ELISA methods (Figures 47-50). Anti-human tissue factor antibody served as the capture antibody and biotinylated anti-human IL-21, IL-15, or TGFβRII served as the detection antibody. Tissue factor was also detected using anti-human tissue factor capture antibody (I43) and anti-human tissue factor antibody detection. The I43/anti-TF antibody ELISA was compared to similar concentrations of purified tissue factor.

Example 27: Immunostimulatory Potential of 21t15-TGFRs Conjugates To assess the IL-15 immunostimulatory activity of 21t15-TGFRs conjugates, increasing concentrations of 21t15-TGFRs were added to 200 μL of IMDM:32Dβ cells in 10% FBS medium. (10 ⁴ cells/well) and cells were incubated at 37° C. for 3 days. On day 4, WST-1 proliferation reagent (10 μL/well) was added and after 4 hours absorbance was measured at 450 nm to measure 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 Figure 51, 21t15-TGFRs exhibited IL-15 dependent 32Dβ cell proliferation. The 21t15-TGFRs complex was reduced compared to human recombinant IL-15, possibly due to binding of IL-21 and tissue factor to the IL-15 domain.

In addition, HEK-Blue TGFβ reporter cells (hkb-tgfb, InvivoGen) were used to measure the ability of 21t15-TGFRs to block TGFβ1 activity (FIG. 52). Increasing concentrations of 21t15-TGFRs were mixed with 0.1 nM TGFβ1 and added to HEK-Blue TGFβ reporter cells (2.5×10 ⁴ cells/well) in 200 μL IMDM:10% heat-inactivated FBS medium. . Cells were incubated overnight at 37°C. The next day, 20 μl of induced HEK-Blue TGFβ reporter cell supernatant was added to 180 μl of QUANTI-Blue (InvivoGen) and incubated at 37° C. for 1-3 hours. 21t15-TGFRs activity was assessed by measuring absorbance at 620 nm. Human recombinant TGFβRII/Fc activity was evaluated as a positive control.

These results indicate that the TGFβRII domain of the 21t15-TGFRs complex retains the ability to capture TGFβ1. The ability of 21t15-TGFRs to block TGFβ1 activity was reduced compared to that of human recombinant TGFβRII/Fc, possibly due to binding to the IL-15Rα sushi domain of TGFβRII.

Example 28 Induction of Cytokine-Induced Memory-Like NK Cells by 21t15-TGFRs Complexes Cytokine- induced memory-like NK cells can be induced ex vivo after overnight stimulation of purified NK cells with saturating amounts of cytokines. These memory-like properties can be measured by increased IL-2 receptor alpha (IL-2Rα, CD25), CD69 (and other activation markers) expression, and IFN-γ production. To assess the ability of 21t15-TGFRs complexes to promote the generation of cytokine-induced memory-like NK cells, purified human NK cells (>95% CD56+) were treated with 1 nM-100 nM of 21t15-TGFRs complexes. time stimulated. 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 a blood bank and CD56+ NK cells were isolated using RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone ) resuspended at 0.2×10 ⁶ /mL in 96-well flat-bottom plates in medium (supplemented). Cells were incubated at 37° C. with either mixed cytokines of hIL-21 (50 ng/ml) (Biolegend) and hIL-15 (50 ng/ml) (NCI), or 21t15-TGFRs complexes at 1 nM, 10 nM, or 100 nM. overnight and 5% CO ₂ 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 (containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). After two washes, cells were analyzed using a BD FACSCelesta™ flow cytometer. (Plotted data-mean fluorescence intensity, Figures 53 and 54).

Fresh human leukocytes were obtained from a blood bank and CD56+ NK cells were isolated using RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone ) resuspended at 0.2 x 106/ml in a 96-well flat-bottom plate in medium (supplemented). Cells were incubated at 37° C. with either mixed cytokines of hIL-21 (50 ng/ml) (Biolegend) and hIL-15 (50 ng/ml) (NCI), or 21t15-TGFRs complexes at 1 nM, 10 nM, or 100 nM. overnight and 5% CO ₂ for 14-18 hours. Cells were then treated with 10 μg/ml Brefeldin A (Sigma) and 1X monensin (eBioscience) 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 (containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes) and fixed at room temperature for 10 minutes. After fixation, cells were washed with 1× permeabilization buffer (eBioscience) (1500 RPM, 5 minutes at room temperature) and stained with intracellular IFN-γ-PE (Biolegend) for 30 minutes at room temperature. Cells were washed again with 1x permeabilization buffer and then with FACS buffer. Cell pellets were resuspended in 300 μl of FACS buffer and analyzed using a BD FACSCelesta™ flow cytometer. (% IFN-γ positive cells plotted, Figure 55).

Example 29: In vitro cytotoxicity of NK cells against human tumor cells Human myeloid leukemia cells K562 (labeled with CellTrace violet) were treated with purified human NK cells (StemCell human NK cells) in the presence of increasing concentrations of 21t15-TGFRs complexes. Cell purification kit (E:T ratio; 2:1) was used). After 20 hours, cultures were harvested, stained with propidium iodide (PI) and evaluated by flow cytometry. As shown in Figure 56, the 21t15-TGFRs complex induced human NK cytotoxicity against K562 compared to controls.

Example 30: Generation of an IL-21/TF Mutant/IL-15 DNA Construct and Resultant Fusion Protein Complex with TGFβRII/IL-15RαSu In a non-limiting example, IL-21 was transformed into tissue factor 219 An IL-21/TF variant/IL-15 DNA construct was generated by directly linking to the N-terminal coding region of the variant and then linking the IL-21/TF variant to the N-terminal coding region of IL-15. .

（ヒトＩＬ－１５）
ＡＡＣＴＧＧＧＴＧＡＡＣＧＴＣＡＴＣＡＧＣＧＡＴＴＴＡＡＡＧＡＡＧＡＴＣＧＡＡＧＡＴＴＴＡＡＴＴＣＡＧＴＣＣＡＴＧＣＡＴＡＴＣＧＡＣＧＣＣＡＣＴＴＴＡＴＡＣＡＣＡＧＡＡＴＣＣＧＡＣＧＴＧＣＡＣＣＣＣＴＣＴＴＧＴＡＡＧＧＴＧＡＣＣＧＣＣＡＴＧＡＡＡＴＧＴＴＴＴＴＴＡＣＴＧＧＡＧＣＴＧＣＡＡＧＴＴＡＴＣＴＣＴＴＴＡＧＡＧＡＧＣＧＧＡＧＡＣＧＣＴＡＧＣＡＴＣＣＡＣＧＡＣＡＣＣＧＴＧＧＡＧＡＡＴＴＴＡＡＴＣＡＴＴＴＴＡＧＣＣＡＡＴＡＡＣＴＣＴＴＴＡＴＣＣＡＧＣＡＡＣＧＧＣＡＡＣＧＴＧＡＣＡＧＡＧＴＣＣＧＧＣＴＧＣＡＡＧＧＡＧＴＧＣＧＡＡＧＡＧＣＴＧＧＡＧＧＡＧＡＡＧＡＡＣＡＴＣＡＡＧＧＡＧＴＴＴＣＴＧＣＡＡＴＣＣＴＴＴＧＴＧＣＡＣＡＴＴＧＴＣＣＡＧＡＴＧＴＴＣＡＴＣＡＡＴＡＣＣＴＣＣ The nucleic acid sequence of the IL-21/TF variant/IL-15 construct (including the signal peptide sequence) is as follows (SEQ ID NO:324, shaded nucleotides are variants, variant codons are underlined). is subtracted).
(signal sequence)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCAGGCTCCGAGGACTCC
(human tissue factor 219 variant)

(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

（ヒトＩＬ－１５）
ＮＷＶＮＶＩＳＤＬＫＫＩＥＤＬＩＱＳＭＨＩＤＡＴＬＹＴＥＳＤＶＨＰＳＣＫＶＴＡＭＫＣＦＬＬＥＬＱＶＩＳＬＥＳＧＤＡＳＩＨＤＴＶＥＮＬＩＩＬＡＮＮＳＬＳＳＮＧＮＶＴＥＳＧＣＫＥＣＥＥＬＥＥＫＮＩＫＥＦＬＱＳＦＶＨＩＶＱＭＦＩＮＴＳ The amino acid sequence (including signal peptide sequence) of the IL-21/TF variant/IL-15 construct is as follows (SEQ ID NO:325, substituted residues are shaded).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(human tissue factor 219)

(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

In some embodiments, the IL-21/TF variant/IL-15 DNA construct is combined with the TGFβRII/IL-15RαSu DNA construct and transfected into cells using a retroviral vector as described above, It can be expressed as an IL-21/TF variant/IL-15 fusion protein and a TGFβRII/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 variant/IL-15 fusion protein to form IL-21/TF variant/IL-15:TGFβRII/ An IL-15RαSu complex is made.

Example 31: Generation of IL-21/IL-15RαSu DNA Constructs and TGFβRII/TF/IL-15 DNA Constructs and Resulting Fusion Protein Complexes In a non-limiting example, IL-21 An IL-21/IL-15RαSu DNA construct was generated by directly linking the unit sequences. The nucleic acid sequence (including signal sequence) of the IL-21/IL-15RαSu construct is as follows (SEQ ID NO:214).
(signal sequence)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

The amino acid sequence (including signal peptide sequence) of the IL-21/IL-15RαSu construct is as follows (SEQ ID NO:213).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

In some embodiments, the IL-21/IL-15RαSu DNA construct is combined with the TGFβRII/TF/IL-15 DNA construct and transfected into a retroviral vector as described above to produce IL-21/IL-15RαSu It can be expressed as a fusion protein and a TGFβRII/TF/IL-15 fusion protein. The IL-15RαSu domain of the IL-21/IL-15RαSu fusion protein binds to the IL-15 domain of the TGFβRII/TF/IL-15 fusion protein to form the TGFβRII/TF/IL-15:IL-21/IL-15RαSu complex. Create a body.

The TGFβRII/TF/IL-15RαSu DNA construct was generated by ligating the TGFβRII sequence to the N-terminal coding region of the human tissue factor 219 form, followed by ligating the TGFβRII/TF construct to the N-terminal coding region of IL-15. A single chain version of TGFβRII (TGFβRII-linker-TGFβRII) was used as described above. The nucleic acid sequence (including leader sequence) of the TGFβRII/TF/IL-15 construct is as follows (SEQ ID NO:239).
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβRII-first fragment)
ATCCCCCCCCCATGTGCCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT
(linker)
GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT
(Human TGFβRII-Second Fragment)
ATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGG TGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCCACGATCCCCAAGCTGCCCTACCCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGC GACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC
(human tissue factor 219)
TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACACAGACACCGAGTGTGATTTAACCGACGAA ATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTTCTACGAGAATTCCCCCGAATTCACCCTTATTTAGAGACCAATTTAGGCCAGCCCTACCATCCAGAGCTTCGAGCCAAGTTGGCACCAAGG TGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCCGGAATAACACATTTTTATCCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAA AGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including signal peptide) of the TGFβRII/TF/IL-15 fusion protein is as follows (SEQ ID NO:238).
(signal peptide)
MKWVT FISLLFLFS SAYS
(Human TGFβRII-first fragment)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPD
(linker)
GGGGSGGGGSGGGGGS
(Human TGFβRII-Second Fragment)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPD
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 32. Production of an Exemplary Single-Chain Chimeric Polypeptide A peptide (αCD3scFv/TF/αCD28scFv) was generated (Figure 57). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.
Nucleic Acid (SEQ ID NO: 158) Encoding an Exemplary Single Chain Chimeric Polypeptide (αCD3scFv/TF/αCD28scFv)
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTTATTCAGCTCCCGCCTATTCC
(αCD3 light chain variable region)
CAGATCGTGCTGACCCAAAGCCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAGGTGGATCTACGACACCAGCAAGCTGGCCTCCGGAGTGCCCG CTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGT
(linker)
GGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGCGAGGAGGAAGC
(αCD3 heavy chain variable region)
CAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGGCTATACATTTACTCGTCATACAATGCATTGGGTCAAGCAGAGGCCCGGTCAAGGTTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGG CTACACCAACTATAACCAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGGAGGACTCCGCTGTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCA GC
(human tissue factor 219 form)
TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAA ATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTTCTACGAGAATTCCCCCGAATTCACCCTTATTTAGAGACCAATTTAGGCCAGCCCTACCATCCAGAGCTTCGAGCCAAGTTGGCACCAAGG TGAACGTCACCGTCGAGGATGAAAAGGACTTTAGTGCGGCCGGAATAACACATTTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAA AGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(αCD28 light chain variable region)
GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTTCACCTCCTATGTGATCCAGTGGGTCAAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACCA AATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGGCGACGGCAATTACTGGGGACGGGGGCCAACACTGACCGTGAGCAGC
(linker)
GGAGGCGGAGGCTCCGGCGGAGGCGCGGATCTGGCGGTGGCGGCTCC
(αCD28 light chain variable region)
GACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCCTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCAATCTCGCCAGCGGCGTG CCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAAACTGGAGACAAAGAGG
Exemplary Single Chain Chimeric Polypeptide (αCD3scFv/TF/αCD28scFv) (SEQ ID NO: 157)
(signal peptide)
MKWVT FISLLFLFSSAYS
(αCD3 light chain variable region)
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR
(linker)
GGGGSGGGGSGGGGGS
(αCD3 heavy chain variable region)
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTTDKSSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(αCD28 light chain variable region)
VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSS
(linker)
GGGGSGGGGSGGGGGS
(αCD28 heavy chain variable region)
DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR

A second exemplary single-chain chimeric polypeptide was generated comprising a first target binding domain that was anti-CD28 scFv, a soluble human tissue factor domain, and a second target binding domain that was anti-CD3 scFv (αCD28scFv/TF /αCD3scFv) (Figure 57). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.
Nucleic Acid (SEQ ID NO:326) Encoding an Exemplary Single Chain Chimeric Polypeptide (αCD28scFv/TF/αCD3scFv)
(signal peptide)
ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCTACAGC
(αCD28 light chain variable region)
GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCCGGTCAAGGTTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACAC CAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGCACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTATACTATTGCGCTCGTTGGGGCGATGGCCAATTATTGGGGCCGGGGGAACTACTTTTAACAGTGAGCTCC
(linker)
GGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGGTGGAGGCAGC
(αCD28 heavy chain variable region)
GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGC CTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCCTACCTTTGGCGCGGAGGCACAAAGCTGGAGACCAAGCGGG
(human tissue factor 219 form)
AGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCAGACACCGAGTGCGATCTCACCGAC GAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCTCTACCCCCGCTGGAAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGG TGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGC GAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGGAGAGTTTCGTGAG
(αCD3 light chain variable region)
CAGATCGTGCTGACCCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCC CCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGT
(linker)
GGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGGTGGAGGTAGC
(αCD3 heavy chain variable region)
ＣＡＡＧＴＴＣＡＧＣＴＣＣＡＧＣＡＡＡＧＣＧＧＣＧＣＣＧＡＡＣＴＣＧＣＴＣＧＧＣＣＣＧＧＣＧＣＴＴＣＣＧＴＧＡＡＧＡＴＧＴＣＴＴＧＴＡＡＧＧＣＣＴＣＣＧＧＣＴＡＴＡＣＣＴＴＣＡＣＣＣＧＧＴＡＣＡＣＡＡＴＧＣＡＣＴＧＧＧＴＣＡＡＧＣＡＡＣＧＧＣＣＣＧＧＴＣＡＡＧＧＴＴＴＡＧＡＧＴＧＧＡＴＴＧＧＣＴＡＴＡＴＣＡＡＣＣＣＣＴＣＣＣＧＧＧＧＣＴＡＴＡＣＣＡＡＣＴＡＣＡＡＣＣＡＧＡＡＧＴＴＣＡＡＧＧＡＣＡＡＡＧＣＣＡＣＣＣＴＣＡＣＣＡＣＣＧＡＣＡＡＧＴＣＣＡＧＣＡＧＣＡＣＣＧＣＴＴＡＣＡＴＧＣＡＧＣＴＧＡＧＣＴＣＴＴＴＡＡＣＡＴＣＣＧＡＧＧＡＴＴＣＣＧＣＣＧＴＧＴＡＣＴＡＣＴＧＣＧＣＴＣＧＧＴＡＣＴＡＣＧＡＣＧＡＴＣＡＴＴＡＣＴＧＣＣＴＣＧＡＴＴＡＣＴＧＧＧＧＣＣＡＡＧＧＴＡＣＣＡＣＣＴＴＡＡＣＡＧＴＣＴＣＣＴＣＣ
Exemplary Single Chain Chimeric Polypeptide (αCD28scFv/TF/αCD3scFv) (SEQ ID NO:327)
(signal peptide)
MKWVT FISLLFLFS SAYS
(αCD28 light chain variable region)
VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSS
(linker)
GGGGSGGGGSGGGGGS
(αCD28 heavy chain variable region)
DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(αCD3 light chain variable region)
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR
(linker)
GGGGSGGGGSGGGGGS
(αCD3 heavy chain variable region)
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTTDKSSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

Nucleic acids encoding 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 allowed secretion of a soluble αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide (termed 3t28), which was assayed by anti-TF antibody affinity and other chromatographic methods. It can be refined.

The αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide was purified using an anti-tissue factor affinity column. The anti-tissue factor affinity column was connected to a GE Healthcare AKTA Avant system. A flow rate of 4 mL/min was used for all steps except the 2 mL/min elution step.

A cell culture harvest containing the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide was adjusted to pH 7.4 with 1 M Tris base and loaded onto an anti-TF antibody affinity column (described above) equilibrated with 5 column volumes of PBS. . After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 by adding 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 kDa. The data in FIG. 58 demonstrate that the anti-tissue factor affinity column can bind to αCD3scFv/TF/αCD28scFv single chain chimeric polypeptides containing a human soluble tissue factor domain. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing.

After each elution, the anti-tissue factor affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 10 column volumes of PBS, 0.05% _NaN3 and stored at 2-8°C.

Analytical size exclusion chromatography (SEC) was performed using a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) connected to an AKTA Avant system (GE Healthcare) to isolate αCD3scFv/TF/αCD28scFv single-chain chimeric poly(s). I did it with a peptide. The column was equilibrated with 2 column volumes of PBS. A flow rate of 0.8 mL/min was used. 200 μL of αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide (1 mg/mL) was injected onto the column using a capillary loop. After injection of the single-chain chimeric polypeptide, 1.25 column volumes of PBS were flushed through the column. The SEC chromatograph is shown in FIG. The data show that there are three protein peaks that likely represent monomeric and dimeric forms of the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide, or other distinct forms.

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 treated with standard sodium dodecyl sulfate polyacrylamide under reducing conditions. Gels (4-12% NuPage Bis-Tris gels) were analyzed by electrophoresis (SDS-PAGE) method. Gels were stained with InstantBlue for approximately 30 minutes and destained overnight in purified water. Figure 60 shows an SDS gel of αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide purified using an anti-tissue factor affinity column. The results show that the purified αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptide has the expected molecular weight (72 kDa) in reducing SDS gels.

Example 33. Functional Characterization of αCD3scFv/TF/αCD28scFv Single Chain Chimeric Polypeptides Formation of αCD3scFv/TF/αCD28scFv single chain chimeric polypeptides was confirmed by an ELISA-based method. αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptides were isolated using an anti-TF antibody (I43)/anti-TF antibody specific ELISA and a capture antibody, an anti-human tissue factor antibody (I43), and a detection antibody, an anti-TF antibody. detected (Fig. 61). A similar concentration of purified tissue factor protein was used as a control.

Further in vitro experiments were performed to determine whether the αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptides were able to activate human peripheral blood mononuclear cells (PBMCs). Fresh human leukocytes were obtained from a blood bank and peripheral blood mononuclear cells (PBMC) were isolated using a density gradient Histopaque (Sigma). Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone ) resuspended at 0.2×10 ⁶ /mL in 96-well flat-bottom plates in medium (supplemented). Cells were stimulated with 0.01 nM to 1000 nM of αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptides for 3 days at 37° C., 5% CO ₂ . 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 (Biolegend) for 30 minutes. After surface staining, cells were washed (1500 RPM at room temperature) in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). 5 minutes). After two washes, cells were resuspended in 300 μL FACS buffer and analyzed by flow cytometry (Celesta-BD Bioscience). The data in Figures 62 and 63 demonstrate that the αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide can stimulate both CD8 ⁺ and CD4 ⁺ T cells. Further experiments were performed to count PBMCs isolated from blood using Histopaque (Sigma) and add 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin ( (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and supplemented with 10% FBS (Hyclone)) at 0.2×10 ⁶ /mL in 96-well flat bottom plates. Cells were then stimulated with 0.01 nM-1000 nM αCD3scFv/TF/αCD28scFv single-chain chimeric polypeptides for 3 days at 37° C., 5% CO ₂ . After 72 hours, cells were harvested and surface stained for CD4-488, CD8-PerCP Cy5.5, CD25-BV421, CD69-APCFire750, CD62L-PE Cy7, and CD44-PE (Biolegend) for 30 minutes. After surface staining, cells were washed (1500 RPM at room temperature) in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). 5 minutes). After two washes, cells were resuspended in 300 μL FACS buffer and analyzed by flow cytometry (Celesta-BD Bioscience). The data again show that the αCD3scFv/TF/αCD28scFv single chain chimeric polypeptide was able to stimulate the activation of CD4 ⁺ T cells (Figure 64).

Example 34: Generation of IL-7/IL-15RαSu DNA Construct In a non-limiting example, an IL-7/IL-15RαSu DNA construct was generated (see Figure 65). Human IL-7, human IL-15RαSu, human IL-15, and human tissue factor 219 sequences were obtained from the UniProt website, and DNA for these sequences was synthesized by Genewiz. A DNA construct was made by joining the IL-7 sequence to the IL-15RαSu sequence. The final IL-7/IL-15RαSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence (including signal peptide sequence) encoding the second chimeric polypeptide of the IL-7/IL-15RαSu construct is as follows (SEQ ID NO:206).
(signal peptide)
ATGGGAGTGAAAGTTCTTTTTGCCCTTTATTGTATTGCTGTGGCCCGAGGCC
(human IL-7)
GATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTAT TCCGTGCTGCTCGCAAGTTGAGGCAATTTCTTAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAGTTTCAGAAGGCCAACAATACTGTTGAACTGCACTGGCCAGGTTAAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGA AGAAAAAAATCTTTAAAGGAACAGAAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC
(Human IL-15Rα sushi domain)
ATCACGTGCCCTCCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTTGAACAAGGCCACGAATGTCGCCCACTGGA CAACCCCCAGTTCTCAAATGCATTAGA

A second chimeric polypeptide (including a signal peptide sequence) of the IL-7/IL-15RαSu construct is as follows (SEQ ID NO:205).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 35: Generation of an IL-21/TF/IL-15 DNA Construct In a non-limiting example, an IL-21 sequence is ligated to the N-terminal coding region of tissue factor 219, and the IL-21/TF construct is further linked to the IL-21/TF construct. An IL-21/TF/IL-15 construct was generated by joining the N-terminal coding region of -15 (Figure 66).

The nucleic acid sequence (including leader sequence) encoding the first chimeric polypeptide of the IL-21/TF/IL-15 construct synthesized by Genewiz is as follows (SEQ ID NO:202).
(signal peptide)
ATGGGAGTGAAAGTTCTTTTTGCCCTTTATTGTATTGCTGTGGCCCGAGGCC
(Human IL-21 fragment)
CAAGGTCAAGATCGCCACATGATTAGAATGCGTCAACTTATAGATATTGTTGATCAGCTGAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAAACTGTGAGTGGTCAGCTTTTTCCTGTTTTCAGAAGGCCCAACTAAAGTCAGCA AATACAGGAAACAATGAAAAGGATAATCAATGTATCAATTAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAGATGATTCATCA GCATCTGTCCCTCTAGAACACACGGAAGTGAAGATTCC
(human tissue factor 219)
TCAGGCACTACAAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCA CCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCTCTCGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGT GGGAACAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAACAGCCAAAACAAAACACTAATGAGTTTTTGATT GATGTGGATAAAGGAGAAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCCGGTAGAGTGTATGGGCCAGGAGAAAAGGGGGAATTCAGAGAA
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

A first chimeric polypeptide of the IL-21/TF/IL-15 construct that includes a leader sequence is SEQ ID NO:201.
(signal peptide)
MGVKVLFALICIAVAEA (SEQ ID NO: 328)
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 36: Secretion of IL-7/IL-15RαSu and IL-21/TF/IL-15 fusion proteins -1 modified retroviral expression vector (as described in Hughes, Hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells resulted in the formation of a soluble IL-21/TF/IL-15:IL-7/IL-15RαSu protein complex (designated 21t15-7s, FIGS. 67 and 68). formation and secretion of The 21t15-7s protein was purified from CHO-K1 cell culture supernatants using anti-TF antibody affinity chromatography and size exclusion chromatography to produce IL-7/IL-15RαSu fusion protein and IL-21/TF/IL- A soluble (non-aggregated) protein complex consisting of 15 fusion proteins was obtained.

In some cases, the leader (signal sequence) peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

Example 37: Purification of 21t15-7s by immunoaffinity chromatography An anti-TF antibody affinity column was connected to a GE Healthcare™ AKTA Avant protein purification system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

The 21t15-7s cell culture harvest was adjusted to pH 7.4 with 1 M Tris base and loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). Absorbance at 280 nm was collected, after which samples were neutralized to pH 7.5-8.0 by the addition of 1 M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon® centrifugal filters with a molecular weight cutoff of 30 KDa. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing.

After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 10 column volumes of PBS, 0.05% sodium azide and stored at 2-8°C.

Example 38: Size Exclusion Chromatography A GE Healthcare Superdex® 200 Increase 10/300 GL gel filtration column was connected to a GE Healthcare AKTA™ Avant protein purification system. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A capillary loop was used to inject 200 μL of 1 mg/mL 7t15-21s conjugate onto the column. Injections were followed with 1.25 column volumes of PBS.

Example 39: SDS-PAGE of 21t15-7s and 21t15-TGFRs
Purified 21t15-7s protein or 21t15-TGFRs protein samples were analyzed using 4-12% NuPage Bis-Tris protein gel SDS-PAGE to determine purity and protein molecular weight. Gels are stained with InstantBlue™ for approximately 30 minutes and then destained overnight in purified water.

Example 40: Glycosylation of 21t15-7s and 21t15-TGFRs in CHO-K1 Cells Glycosylation of 21t15-7s in CHO-K1 cells or 21t15-TGFRs in CHO-K1 cells was performed using the Protein Deglycosylation Mix II kit (New England Biolabs ) was used according to the manufacturer's instructions.

Example 41: Recombinant protein quantification of 21t15-7s and 21t15-TGFRs complexes 21t15-7s complexes or 21t15-TGFRs complexes were detected and quantified using standard sandwich ELISA methods. Anti-human tissue factor antibody (IgG1) served as a capture antibody, biotinylated anti-human IL-21, IL-15, or IL-7 antibody (21t15-7s), or biotinylated anti-human IL-21, IL-15 , or TGF-βRII antibodies (21t15-TGFRs) functioned as detection antibodies. Tissue factor in the purified 21t15-7s protein complex or 21t15-TGFRs protein complex was detected using an anti-human tissue factor capture antibody and an anti-human tissue factor antibody (IgG1) detection antibody. The anti-TF antibody ELISA is compared to similar concentrations of purified tissue factor.

Example 42: Generation of IL-21/IL-15RαSu DNA Constructs In a non-limiting example, IL-21/IL-15RαSu DNA constructs were generated. Human IL-21 and human IL-15RαSu sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made by joining the IL-21 sequence to the IL-15RαSu sequence. The final IL-21/IL-15RαSu DNA construct sequence was synthesized by Genewiz. See FIG.

Example 43: Generation of an IL-7/TF/IL-15 DNA Construct In a non-limiting example, an IL-7 sequence is ligated to the N-terminal coding region of Tissue Factor 219 and the IL-7/TF construct is further linked to the IL-7/TF construct. The IL-7/TF/IL-15 construct was made by joining the N-terminal coding region of -15. See FIG.

Example 44: Generation of IL-21/IL-15Rα Sushi DNA Constructs In a non-limiting example, a second chimeric polypeptide of IL-21/IL-15RαSu was generated. Human IL-21 and human IL-15Rα sushi sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. A DNA construct was made by joining the IL-21 sequence to the IL-15Rα sushi sequence. The final IL-21/IL-15RαSu DNA construct sequence was synthesized by Genewiz.

The nucleic acid sequence (including leader sequence) encoding the second chimeric polypeptide of the IL-21/IL-15RαSu domain synthesized by Genewiz is as follows (SEQ ID NO:214).
(signal peptide)
ATGAAGTGGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

A second chimeric polypeptide (including a leader sequence) of the IL-21/IL-15Rα sushi domain is as follows (SEQ ID NO:213).
(signal sequence)
MKWVT FISLLFLFSSAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

Example 45: Generation of an IL-7/TF/IL-15 DNA Construct In a non-limiting example, an IL-7 sequence is ligated to the N-terminal coding region of Tissue Factor 219 and the IL-7/TF construct is further linked to the IL-7/TF construct. An exemplary first chimeric polypeptide of IL-7/TF/IL-15 was generated by joining the N-terminal coding region of -15. The nucleic acid sequence (including leader sequence) encoding the IL-7/TF/IL-15 first chimeric polypeptide synthesized by Genewiz is as follows (SEQ ID NO:210).
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human IL-7 fragment)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

A first chimeric polypeptide (including a leader sequence) of IL-7/TF/IL-15 is as follows (SEQ ID NO:209).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Example 46: Secretion of IL-21/IL-15RαSu and IL-7/TF/IL-15 fusion proteins -1 modified retroviral expression vector (as described in Hughes, Hum Gene Ther 16:457-72, 2005, incorporated herein by reference) and the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allows formation and secretion of a soluble IL-7/TF/IL-15:IL-21/IL-15RαSu protein complex (termed 7t15-21s). Became. The 7t15-21s protein was purified from CHO-K1 cell culture supernatants using anti-TF antibody (IgG1) affinity chromatography and size exclusion chromatography, and the IL-21/IL-15RαSu fusion protein and IL-7/ A soluble (non-aggregated) protein complex consisting of the TF/IL-15 fusion protein was obtained. See Figures 71 and 72.

Example 47: Ability to Proliferate Primary Natural Killer (NK) Cells by 7t15-21s Complex + Anti-TF IgG1 Antibody The 21s conjugate and 7t15-21s conjugate plus anti-TF IgG1 antibody are added to NK cells obtained from fresh human leukocyte samples. Cells are stimulated with 50 nM 7t15-21s complex with or without 25 nM anti-TF IgG1 or anti-TF IgG4 antibodies at 37° C. and 5% CO ₂ . Cells are maintained at a concentration of 0.5×10 ⁶ /mL to less than 2.0× ^{10 6} /mL by counting every 48-72 hours and medium is supplemented with fresh stimulant. Cells stimulated with 7t15-21s conjugate or anti-TF IgG1 antibody or anti-TF IgG4 antibody or anti-TF IgG4 plus 7t15-21s conjugate are maintained for up to 5 days. Proliferation of primary NK cells upon incubation with 21t15-7s complex plus anti-TF IgG1 antibody is observed.

Example 48: Activation of Expanded NK Cells by 7t15-21s Conjugate + Anti-TF IgG1 Antibody Primary NK cells are induced ex vivo after overnight stimulation of purified NK cells with 7t15-21s conjugate + anti-TF IgG1 antibody can do. Fresh human leukocytes are obtained from a blood bank and CD56+ NK cells are isolated using RosetteSep/human NK cell reagent (StemCell Technologies). NK cell purity is >80% and is confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). Cells were counted and added to 1 mL complete medium (RPMI 1640 (Gibco), 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acids (Thermo Life T technologies), pyruvin Resuspend at 1×10 ⁶ /mL in 24-well flat-bottom plates in sodium phosphate (Thermo Life Technologies), and supplemented with 10% FBS (Hyclone). Cells are stimulated with 50 nM 7t15-21s with or without 25 nM anti-TF IgG1 antibody at 37° C. and 5% CO ₂ . Cells are counted every 48-72 hours and maintained at a concentration of 0.5×10 ⁶ /mL to 2.0× ^{10 6} /mL until day 14. The medium is regularly replenished with fresh stimulants. Cells are harvested and on day 3 surface stained with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 specific antibodies (Biolegend) and analyzed by Flow Cytometry-Celeste-BD Bioscience. MFI is observed to increase with 7t15-21s complex + anti-TF IgG1 antibody stimulation, but not with 7t15-21s complex stimulation The activation marker CD69 MFI is observed to increase with 7t15-21s complex + anti-TF IgG1 antibody stimulation and the 7t15-21s complex alone are observed to increase.

Example 49: Increased Glucose Metabolism of NK Cells Using 18t15-12s A series of experiments were performed demonstrating the effect of the 18t15-12s construct on the rate of oxygen consumption and extracellular acidification (ECAR) of NK cells purified from human blood. It was determined.

In these experiments, fresh human leukocytes were obtained from blood banks from two different human donors and NK cells were isolated by negative selection using RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >80% and confirmed by staining with CD56-BV421 and CD16-BV510 specific antibodies (BioLegend). Cells were counted and added to 1 mL complete medium (RPMI 1640 (Gibco), 4 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), non-essential amino acids (Thermo Life T technologies), pyruvin resuspended at 2×10 ⁶ /mL in 24-well flat-bottom plates in sodium phosphate (Thermo Life Technologies), and supplemented with 10% FBS (Hyclone). Cells were treated with (1) media alone, (2) 100 nM 18t15-12s, or (3) the single cytokines recombinant human IL-12 (0.25 μg), recombinant human IL-15 (1.25 μg). and recombinant human IL-18 (1.25 μg) overnight at 37° C., 5% CO ₂ . The next day, cells were harvested and an extracellular flux assay of expanded NK cells was performed using an XFp analyzer (Seahorse Bioscience). Harvested cells were washed and plated at 2.0×10 ⁵ cells/well at least in duplicate for extracellular flux analysis of OCR (oxygen consumption rate) and ECAR (extracellular acidification rate). Glycolytic stress tests were performed in Seahorse medium containing 2 mM glutamine. The following were used in the assay: 10 mM glucose, 100 nM oligomycin, and 100 mM 2-deoxy-D-glycose (2DG).

The data show that 18t15-12s compared to the same cells activated with a combination of recombinant human IL-12, recombinant human IL-15, and recombinant human IL-18 increased the oxygen consumption rate (Figure 73). and markedly increased the extracellular acidification rate (ECAR) (Figure 74).

Example 50 Generation and Characterization of 7t15-16s21 Fusion Protein A fusion protein complex consisting of an anti-CD16scFv/IL-15RαSu/IL-21 fusion protein and an IL-7/TF/IL-15 fusion protein was generated. Human IL-7 and human IL-21 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made by joining the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of a construct containing IL-7 attached to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the IL-7/TF/IL-15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-7)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the IL-7/TF/IL-15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

A construct was also made by joining the anti-CD16 scFv sequence to the N-terminal coding region of the IL-15RαSu chain synthesized by Genewiz, followed by the N-terminal coding region of IL-21. The nucleic acid and protein sequences of a construct containing anti-CD16 scFv attached to the N-terminus of IL-15RαSu chain followed by the N-terminal coding region of IL-21 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the anti-CD16SscFv/IL-15RαSu/IL-21 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
((anti-human CD16scFv)
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG TTCTCCGGATCCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGGCGGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTGTCCGGAGGCGGCCG GCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAGGGGCCTGGAGTGGGTGT CCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGA CAGGGCACCCTGGTGACCGTGTCCCAGG
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence (including signal peptide sequence) of the anti-CD16 scFv/IL-15RαSu/IL-21 construct is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(Anti-human CD16scFv)
SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGSGGGGSEVQLVESGGGGVVRPGG SLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The anti-CD16 scFv/IL-15RαSu/IL-21 and IL-7/TF/IL-15 constructs were cloned into modified retroviral expression vectors 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 Ther2005 16:457-72), and the expression vector was transfected into CHO-K1 cells. . Co-expression of the two constructs in CHO-K1 cells yielded a soluble IL-7/TF/IL-15:anti-CD16scFv/IL-15RαSu/IL-21 protein complex (designated 7t15-16s21, FIGS. 75 and 76). ), which can be purified by anti-TF IgG1 antibody-based affinity and other chromatographic methods.

Binding of 7t15-16s21 to CHO Cells Expressing Human CD16b CHO cells were transfected with human CD16b in the pMC plasmid and selected with 10 μg/mL blasticidin for 10 days. CHO cells stably expressing CD16b were stained with 7t15-16s21 containing 1.2 μg/mL anti-human CD16 scFv or 18t15-12s without anti-human CD16 scFv as a negative control followed by biotinylated anti-human tissue factor. and stained with PE-conjugated streptavidin. As shown in Figure 77A, only 7t15-16s21 containing anti-human CD16 scFv stained cells. As shown in Figure 77B, 18t15-12s did not stain CHO cells expressing human CD16b.

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 treated at room temperature (RT ) for 2 hours. Plates were washed three times and blocked with 100 μL of 1% BSA in PBS. Serial dilutions of 7t15-16s21 (1:3 ratio) were added to the wells and incubated for 60 minutes at room temperature. After three washes, 50 ng/mL biotinylated anti-IL15 antibody (BAM247, R&D Systems), 500 ng/mL biotinylated anti-IL-21 antibody (13-7218-81, R&D Systems), or 500 ng/mL biotin. Anti-IL-7 antibody (506602, R&D Systems) was incubated with the wells for 60 minutes at room temperature. Plates were washed 3 times and incubated with 0.25 μg/mL HRP-SA (Jackson ImmunoResearch) at 100 μL/well for 30 minutes at room temperature, then washed 4 times and incubated with 100 μl ABTS for 2 minutes at room temperature. . Absorbance was read at 405 nm. As shown in Figures 78A-78C, the IL-15, IL-21, and IL-7 domains in 7t15-16s21 were detected by individual antibodies.

IL-15 at 7t15-16s21 Promotes Proliferation of IL-2Rβ and Common γ Chain Containing 32Dβ Cells To analyze IL-15 activity at 7t15-16s21, IL-15 activity at 7t15-16s21 and 32Dβ cells expressing the common γ chain were used to compare recombinant IL-15 and assess their effects on promoting cell proliferation. IL-15 dependent 32Dβ cells were washed 5 times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serially diluted 7t15-16s21 or IL-15 were added to the cells (Figure 79). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μl of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ incubator. Absorbance was measured at 450 nm by analyzing the amount of formazan dye produced. As shown in Figure 79, 7t15-16s21 and IL-15 promoted 32Dβ cell proliferation and the _EC50 of 7t15-16s21 and IL-15 were 172.2 pM and 16.63 pM, respectively.

Purification Elution Chromatography of 7t15-16s21 from Anti-TF Antibody Affinity Column 7t15-16s21 harvested from cell culture was 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 and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. FIG. 80 is a line graph showing the chromatographic profile of 7t15-16s21 protein-containing cell culture supernatant after binding to and elution on anti-TF antibody resin. As shown in Figure 80, the anti-TF antibody affinity column bound 7t15-16s21 containing TF. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Analytical Size Exclusion Chromatography (SEC) Analysis of 7t15-16s21 To perform size exclusion chromatography (SEC) analysis of 7t15-16s21, a Superdex200 Increase 10/300 GL gel filtration column ( GE Healthcare) was used. The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing 7t15-16s21 in PBS was injected onto the Superdex200 column using a capillary loop and analyzed by SEC. As shown in Figure 81, the SEC results showed two protein peaks of 7t15-16s21.

Example 51: Generation and Characterization of TGFRt15-16s21 Fusion Protein A fusion protein complex comprising anti-human CD16scFv/IL-15RαSu/IL21 and a TGFβ receptor II/TF/IL-15 fusion protein was generated (FIGS. 82 and Figure 83). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a G4S(3) linker was used to join two TGFβ receptor II sequences to generate a single-chain version of TGFβ receptor II, followed by the N-terminal coding region of tissue factor 219 followed by A construct was made by directly linking to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of a construct containing two TGFβ receptor II bound 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 TGFβ receptor II/TF/IL-15 constructs are as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(two human TGFβ receptor II fragments)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the TGFβ receptor II/TF/IL-15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly linking the anti-human CD16 scFv to the N-terminal coding region of the IL-15RαSu chain synthesized by Genewiz, followed by the N-terminal coding region of IL-21. The nucleic acid and protein sequences of a construct containing anti-human CD16 scFv attached 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 signal peptide sequence) of the anti-CD16 scFv/IL-15RαSu/IL-21 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Anti-human CD16scFv)
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG TTCTCCGGATCCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGGCGGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTCCGGAGGCGGCCG GCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGGCTCCTGGAAAGGGGCCTGGAGTGGGTGT CCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGA CAGGGCACCCTGGTGACCGTGTCCCAGG
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC

The amino acid sequence (including signal peptide sequence) of the anti-CD16 scFv/IL-15RαSu/IL-21 construct is as follows.
(signal peptide)
MKWVT FISLLFLFSSAYS
(anti-human CD16scFv)
SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGSGGGGSEVQLVESGGGGVVRPGG SLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The anti-CD16 scFv/IL-15RαSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors 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 Ther2005;1 6:457-72), the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allows formation and secretion of a soluble TGFR/TF/IL-15:CD16scFv/IL-15RαSu/IL-21 protein complex (termed TGFRt15-16s21). , which can be purified by anti-TF IgG1-based affinity and other chromatographic methods.

Interaction between TGFRt15-16s21 and CHO cells expressing human CD16b CHO cells were transfected with human CD16b in the pMC plasmid and selected with 10 μg/mL blasticidin for 10 days. Cells stably expressing CD16b were stained with TGFRt15-16s21 containing 1.2 μg/mL anti-human CD16 scFv or 7t15-21s without anti-human CD16 scFv as a negative control, biotinylated anti-human tissue factor antibody and PE. Stained with conjugated streptavidin. As shown in Figures 84A and 84B, TGFRt15-16s21 containing anti-human CD16 scFv showed positive binding, while 7t15-21s showed no binding.

Effect of TGFRt15-16s21 on TGFβ1 Activity in HEK-Blue TGFβ Cells To assess the activity of TGFβRII in TGFRt15-16s21, the effect of TGFRt15-16s21 on TGFβ1 activity in HEK-Blue TGFβ cells was analyzed. HEK-Blue TGFβ cells (Invivogen) were washed twice with prewarmed PBS and plated 5× in test medium (DMEM, 10% heat-inactivated FCS, 1× glutamine, 1× anti-anti, and 2× glutamine). Resuspend at 10 ⁵ cells/mL. In a flat bottom 96-well plate, 50 μL of cells were added to each well (2.5×10 ⁴ cells/well) followed by 50 μl of 0.1 nM TGFβ1 (R&D systems). TGFRt15-16s21 or TGFR-Fc (R&D Systems) prepared in 1:3 serial dilutions were then added to the plate to a total volume of 200 μL. After 24 hours of incubation at 37°C, 40 μL of induced HEK-Blue TGFβ cell supernatant is added to 160 μL of prewarmed QUANTI-Blue (Invivogen) in a flat bottom 96-well plate and incubated for 1-3 hours at 37°C. bottom. OD values were then determined at 620-655 nM using a plate reader (Multiscan Sky). The _IC50 for each protein sample was calculated using GraphPad Prism 7.04. The IC ₅₀ for TGFRt15-16s21 and TGFR-Fc were 9127 pM and 460.6 pM, respectively. These results indicated that the TGFβRII domain in TGFRt15-16s21 was able to block TGFβ-1 activity in HEK-Blue TGFβ cells.

IL-15 at TGFRt15-16s21 Promotes Proliferation of IL-2Rβ and Common γ Chain-Containing 32Dβ Cells To analyze the activity of IL-15 at TGFRt15-16s21, IL-15 activity of and 32Dβ cells expressing the common γ chain were used to compare recombinant IL-15 and assess their effects on promoting cell proliferation. IL-15 dependent 32Dβ cells were washed 5 times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serial dilutions of TGFRt15-16s21 or IL-15 were added to the cells (Figure 86). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μL of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ incubator. Absorbance was measured at 450 nm by analyzing the amount of formazan dye produced. Data are shown in FIG. As shown in Figure 86, TGFRt15-16s21 and IL-15 stimulated 32Dβ cell proliferation and the _EC50 of TGFRt15-16s21 and IL-15 were 51298 pM and 10.63 pM, respectively.

Detection of IL-15, IL-21 and TGFβRII 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 at room temperature (RT). Incubated for 2 hours. Plates were washed three times and blocked with 100 μL of 1% BSA in PBS. TGFRt15-16s21 serially diluted in a 1:3 ratio was added and incubated for 60 minutes at room temperature. After three washes, 50 ng/mL biotinylated anti-IL-15 antibody (BAM247, R&D Systems), 500 ng/mL biotinylated anti-IL-21 antibody (13-7218-81, R&D Systems), or 200 ng/mL of biotinylated anti-TGFβRII antibody (BAF241, R&D Systems) was applied per well and incubated for 60 minutes at room temperature. After 3 washes, 0.25 μg/mL HRP-SA (Jackson ImmunoResearch) was incubated with 100 μL/well for 30 minutes at room temperature, followed by 4 washes and incubation with 100 μL ABTS for 2 minutes at room temperature. was read at 405 nm, IL-15, IL-21, and TGFβRII domains in TGFRt15-16s21 were detected by the respective antibodies, as shown in Figures 87A-87C.

Purification Elution Chromatography of TGFRt15-16s21 Using Anti-TF Antibody Affinity Column TGFRt15-16s21 collected from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. As shown in Figure 88, the anti-TF antibody affinity column bound TGFRt15-16s21 with tissue factor as a fusion partner. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Reducing SDS-PAGE of TGFRt15-16s21
To determine the purity and molecular weight of the TGFRt15-16s21 protein, anti-TF antibody affinity column purified protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis under reducing conditions ( analyzed by SDS-PAGE). After electrophoresis, gels were stained with InstantBlue for approximately 30 minutes and then destained overnight in purified water.

To verify that the TGFRt15-16s21 protein undergoes post-translational glycosylation in CHO cells, deglycosylation experiments were performed using the Protein Deglycosylation Mix II kit from New England Biolabs according to the manufacturer's instructions. FIG. 89 shows the results of reducing SDS-PAGE analysis of samples in the non-deglycosylated state (lane 1 in red border) and deglycosylated state (lane 2 in yellow border). The results showed that the TGFRt15-16s21 protein was glycosylated when expressed in CHO cells. After deglycosylation, purified samples showed the expected molecular weights (69 kDa and 48 kDa) in reducing SDS gels. Lane M was loaded with 10 μL of SeeBlue Plus2 Prestained Standard.

Example 52 Generation and Characterization of 7t15-7s Fusion Proteins Fusion protein complexes containing IL-7/TF/IL-15 fusion proteins and IL-7/IL-15RαSu fusion proteins were generated (FIGS. 90 and 91) ). Human IL-7, tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made by joining the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of a construct containing IL-7 attached to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the 7t15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL7)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the 7t15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

A construct was also made by joining the IL-7 sequence to the N-terminal coding region of the IL-15RαSu chain synthesized by Genewiz. The nucleic acid and protein sequences of constructs containing IL-7 attached to the N-terminus of IL-15RαSu chain are shown below.

The nucleic acid sequence of the 7s construct (including signal peptide sequence) is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL7)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

The amino acid sequence (including leader sequence) of the 7s fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The IL-7/TF/IL-15 and IL-7/IL-15RαSu constructs were cloned into modified retroviral expression vectors previously described (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. 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 vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allowed the formation and secretion of a soluble IL-7/TF/IL-15:IL-7/IL-15RαSu protein complex termed 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 harvested from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. As shown in Figure 92, the anti-TF antibody affinity column bound 7t15-7s with tissue factor (TF) as a fusion partner. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Immunostimulation of 7t15-7s in C57BL/6 Mice -7 and a second polypeptide that is a soluble fusion (7s) of the sushi domain of the human IL-15 receptor alpha chain (a type A multichain polypeptide described herein) be.

CHO cells were co-transfected with IL7-TF-IL-15 (7t15) and IL7-IL-15Ra sushi domain (7s) vectors. The 7t15-7s complex was purified from transfected CHO cell culture supernatants. IL-7, IL-15, and tissue factor (TF) components in the complex were demonstrated by ELISA, as shown in FIG. Humanized anti-TF antibody monoclonal antibody (anti-TF IgG1) was used as capture antibody to determine TF in 7t15-7s, biotinylated anti-human IL-15 antibody (R&D systems) and biotinylated anti-human IL-7 Antibodies (R&D Systems) were used as detection antibodies to detect IL-15 and IL-7 in 7t15-7s, respectively, followed by peroxidase-conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS substrate (Surmodics IVD, Inc.). .)It was used.

7t15-7s was injected subcutaneously into C57BL/6 mice at 10 mg/kg to determine the immunostimulatory activity of 7t15-7s in vivo. C57BL/6 mice treated subcutaneously with PBS were used as controls. Mouse spleens were harvested and weighed 4 days after treatment. Single splenocyte suspensions were prepared and analyzed by flow cytometry for percentages of CD4 ⁺ T cells, CD8 + T cells, and NK cells with fluorochrome-conjugated anti-CD4, anti ^- CD8, and anti-NK1.1 antibodies. . The results showed that 7t15-7s was effective in expanding splenocytes based on spleen weight (FIG. 94A), specifically the percentage of CD8 ⁺ T cells and NK cells increased compared to control treated mice. was higher (Fig. 94B).

Example 53: Generation and Characterization of TGFRt15-TGFRs Fusion Proteins A fusion protein complex consisting of a TGFβ receptor II/IL-15RαSu fusion protein and a TGFβ receptor II/TF/IL-15 fusion protein was generated (FIGS. 95 and Figure 96). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a G4S(3) linker was used to join two TGFβ receptor II sequences to generate a single-chain version of TGFβ receptor II, followed by the N-terminal coding region of tissue factor 219 followed by A construct was made by directly ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of a construct containing two TGFβ receptor II bound 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 TGFβ receptor II/TF/IL-15 constructs are as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(two human TGFβ receptor II fragments)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the TGFβ receptor II/TF/IL-15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly coupling two TGFβ receptor II to IL-15RαSu chains synthesized by Genewiz. The nucleic acid and protein sequences of a construct containing TGFβ receptor II attached to the N-terminus of IL-15RαSu are shown below.

The nucleic acid sequence (including signal peptide sequence) of the TGFβ receptor II/IL-15RαSu construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(two human TGFβ receptor II fragments)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

The amino acid sequences (including signal peptide sequences) of the two TGFβ receptor II/IL-15RαSu constructs are as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(two human TGFβ receptor II extracellular domains)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The TGFβR/IL-15RαSu and TGFβR/TF/IL-15 constructs were cloned into the modified retroviral expression vector 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; CHO-K1 cells were transfected. Co-expression of these two constructs in CHO-K1 cells allowed the formation and secretion of soluble TGFβR/TF/IL-15:TGFβR/IL-15RαSu protein complexes (termed TGFRt15-TGFRs), which can be purified by anti-TF IgG1 affinity chromatography and other chromatographic methods.

Effect of TGFRt15-TGFRs on TGFβ1 Activity in HEK-Blue TGFβ Cells To assess the activity of TGFβRII in TGFRt15-16s21, the effect of TGFRt15-TGFRs on TGFβ1 activity in HEK-Blue TGFβ cells was analyzed. HEK-Blue TGFβ cells (Invivogen) were washed twice with prewarmed PBS and plated 5× in test medium (DMEM, 10% heat-inactivated FCS, 1× glutamine, 1× anti-anti, and 2× glutamine). Resuspend at 10 ⁵ cells/mL. In a flat bottom 96-well plate, 50 μL of cells were added to each well (2.5×10 ⁴ cells/well) followed by 50 μL of 0.1 nM TGFβ1 (R&D systems). TGFRt15-16s21 or TGFR-Fc (R&D Systems) prepared in 1:3 serial dilutions were then added to the plate to a total volume of 200 μL. After 24 hours of incubation at 37°C, 40 μL of induced HEK-Blue TGFβ cell supernatant is added to 160 μL of prewarmed QUANTI-Blue (Invivogen) in a flat bottom 96-well plate and incubated for 1-3 hours at 37°C. bottom. OD values were then determined at 620-655 nM using a plate reader (Multiscan Sky) (Figure 97). The _IC50 for each protein sample was calculated using GraphPad Prism 7.04. The IC ₅₀ for TGFRt15-TGFRs and TGFR-Fc were 216.9 pM and 460.6 pM, respectively. These results indicated that the TGFβRII domain in TGFRt15-TGFRs could block TGFβ1 activity in HEK-Blue TGFβ cells.

IL-15 in TGFRt15-TGFRs Promotes Proliferation of IL-2Rβ and Common γ Chain Containing 32Dβ Cells , IL2Rβ and 32Dβ cells expressing the common γ chain were used to compare recombinant IL-15 and assess their effects on promoting cell proliferation. IL-15 dependent 32Dβ cells were washed 5 times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serially diluted TGFRt15-TGFRs or IL-15 were added to the cells (Figure 98). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μL of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ incubator. Absorbance was measured at 450 nm by analyzing the amount of formazan dye produced. As shown in Figure 98, TGFRt15-TGFRs and IL-15 promoted 32Dβ cell proliferation, with _EC50s of TGFRt15-16s21 and IL-15 of 1901 pM and 10.63 pM, respectively.

Detection of IL-15 and TGFβRII domains in TGFRt15-TGFRs using corresponding antibodies using ELISA A 96-well plate was coated with 100 μL (8 μg/mL) of anti-TF IgG1 in R5 (coating buffer) and incubated at room temperature. (RT) for 2 hours. Plates were washed three times and blocked with 100 μL of 1% BSA in PBS. TGFRt15-TGFRs were added at 1:3 serial dilutions and incubated for 60 minutes at room temperature. After three washes, 50 ng/mL biotinylated anti-IL-15 antibody (BAM247, R&D Systems) or 200 ng/mL biotinylated anti-TGFbRII antibody (BAF241, R&D Systems) is added to the wells and incubated for 60 minutes at room temperature. bottom. Plates were then washed 3 times and 0.25 μg/mL HRP-SA (Jackson ImmunoResearch) was added at 100 μL/well and incubated at room temperature for 30 minutes, followed by 4 washes and 100 μL ABTS at room temperature. Incubate for 2 minutes. Absorbance was read at 405 nm. As shown in Figures 99A and 99B, the IL-15 and TGFβRII domains in TGFRt15-TGFRs were detected by individual antibodies.

Purification Elution Chromatography of TGFRt15-TGFRs from Anti-TF Antibody Affinity Column TGFRt15-TGFRs collected from cell culture were loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. As shown in Figure 100, the anti-TF antibody affinity column bound TGFRt15-TGFRs containing TF as a fusion partner. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Analytical Size Exclusion Chromatography (SEC) Analysis of TGFRt15-TGFRs A Superdex200 Increase 10/300 GL gel filtration column (GE Healthcare) was connected to an AKTA Avant system (GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. A sample containing TGFRt15-TGFRs in PBS was injected onto a Superdex200 column using a capillary loop and analyzed by SEC. An SEC chromatograph of the sample is shown in FIG. SEC results showed four protein peaks of TGFRt15-TGFRs.

Reducing SDS-PAGE Analysis of TGFRt15-TGFRs To determine the purity and molecular weight of TGFRt15-TGFRs proteins, anti-TF antibody affinity column-purified protein samples were run on sodium dodecyl sulfate polyacrylamide gels (4-12%) under reducing conditions. were analyzed by NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method. After electrophoresis, gels were stained with InstantBlue for approximately 30 minutes and then destained overnight in purified water.

To verify that the TGFRt15-TGFRs proteins undergo post-translational glycosylation in CHO cells, deglycosylation experiments were performed using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer's instructions. FIG. 102 shows reducing SDS-PAGE analysis of samples in the non-deglycosylated state (lane 1 in red border) and deglycosylated state (lane 2 in yellow border). The results showed that the TGFRt15-TGFRs protein was glycosylated when expressed in CHO cells. After deglycosylation, purified samples showed the expected molecular weights (69 kDa and 39 kDa) in reducing SDS gels. Lane M was loaded with 10ul of SeeBlue Plus 2 Prestained Standard.

Immunostimulatory Activity of TGFRt15-TGFRs in C57BL/6 Mice and a second polypeptide that is a soluble fusion of the sushi domain of the human IL-15 receptor alpha chain (a type A multichain polypeptide described herein).

Wild-type C57BL/6 mice were treated subcutaneously with either control solution or TGFRt15-TGFRs at dosages of 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg. After 4 days of treatment, spleen weight and percentage of various immune cell types present in the spleen were assessed. As shown in FIG. 103A, spleen weight in mice treated with TGFRt15-TGFRs increased with increasing dosage of TGFRt15-TGFRs. In addition, spleen weights in mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs, respectively, were higher compared to mice treated with control solution. In addition, the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, NK cells, and CD19 ⁺ B cells present in the spleens of control-treated and TGFRt15-TGFRs-treated mice was assessed. As shown in FIG. 103B, in the spleen of mice treated with TGFRt15-TGFRs, the percentage of both CD8 ⁺ T cells and NK cells increased with increasing dosage of TGFRt15-TGFRs. Specifically, the percentage of CD8 ⁺ T cells was higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs compared to control-treated mice, and the percentage of NK cells was higher. was higher in mice treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs compared to control-treated mice. These results indicate that TGFRt15-TGFRs can stimulate immune cells in the spleen, especially CD8 ⁺ T cells and NK cells.

The pharmacokinetics of TGFRt15-TGFRs molecules were evaluated in wild-type C57BL/6 mice. Mice were treated subcutaneously with TGFRt15-TGFRs at a dosage of 3 mg/kg. Mouse blood was collected from the tail vein at various time points and serum was prepared. TGFRt15-TGFRs concentrations in mouse sera were measured using ELISA (capture: anti-human tissue factor antibody, detection: biotinylated anti-human TGFβ receptor antibody followed by peroxidase-conjugated streptavidin and ABTS substrate). Results showed that the half-life of TGFRt15-TGFRs in C57BL/6 mice was 12.66 hours.

Mouse splenocytes were prepared to evaluate the immunostimulatory activity of TGFRt15-TGFRs in mice over time. As shown in Figure 104A, spleen weights of mice treated with TGFRt15-TGFRs increased at 48 hours after treatment and continued to increase over time. In addition, the percentage of CD4 ⁺ T cells, CD8 ⁺ T cells, NK cells, and CD19 ⁺ B cells present in the spleens of control-treated and TGFRt15-TGFRs-treated mice was assessed. As shown in FIG. 104B, in the spleens of mice treated with TGFRt15-TGFRs, the percentages of both CD8 ⁺ T cells and NK cells increased at 48 hours after treatment and increased over time after single dose treatment. rice field. These results further demonstrate that TGFRt15-TGFRs can stimulate immune cells in the spleen, especially CD8 ⁺ T cells and NK cells.

Furthermore, dynamic proliferation of immune cells based on splenocyte Ki67 expression and cytotoxic potential based on granzyme B expression were examined in splenocytes isolated from mice after a single dose of TGFRt15-TGFRs (3 mg/kg). evaluated. As shown in Figures 105A and 105B, in the spleen of mice treated with TGFRt15-TGFRs, NK cell expression of Ki67 and granzyme B was increased at 24 hours post-treatment, and CD8 ⁺ T cell and NK cell expression was Both increased at 48 hours and later after single dose treatment. These results indicate that TGFRt15-TGFRs not only increase the number of CD8 ⁺ T cells and NK cells, but also enhance the cytotoxicity of these cells. A single dose treatment of TGFRt15-TGFRs proliferated CD8 ⁺ T cells and NK cells for at least 4 days.

Cytotoxicity of splenocytes from TGFRt15-TGFRs treated mice against tumor cells was also assessed. Mouse Moloney leukemia cells (Yac-1) were labeled with CellTrace Violet and used as tumor target cells. Splenocytes were prepared from mouse spleens treated with TGFRt15-TGFRs (3 mg/kg) at various time points after treatment and used as effector cells. Target cells were mixed with effector cells at an E:T ratio of 10:1 and incubated at 37° C. for 20 hours. Target cell viability was assessed by analysis of propidium iodide-positive violet-labeled Yac-1 cells using flow cytometry. Percentage of Yac-1 tumor inhibition was calculated using the formula (1−[number of viable Yac-1 cells in experimental sample]/[number of viable Yac-1 cells in sample without splenocytes])×100. bottom. As shown in Figure 106, splenocytes from mice treated with TGFRt15-TGFRs had greater cytotoxicity against Yac-1 cells than control mouse splenocytes.

Tumor Size Analysis in Response to Chemotherapy and/or TGFRt15-TGFRs Pancreatic cancer cells (SW1990, ATCC® CRL-2172) were transferred to C57BL/6 scid mice (The Jackson Laboratory, 001913, 2×10 ⁶ cells/mouse, 100 μL). in HBSS) to establish a pancreatic cancer mouse model. Two weeks after tumor cell injection, these mice received chemotherapy with a combination of Abraxane (Celgene, 68817-134, 5 mg/kg, ip) and gemcitabine (Sigma Aldrich, G6423, 40 mg/kg, ip). Immunotherapy with TGFRt15-TGFRs (3 mg/kg, s.c.) was initiated ip at 2 days later. The above procedure was considered one treatment cycle and was repeated for 3 more cycles (1 cycle/week). Control groups were set up as SW1990-injected mice receiving PBS, chemotherapy (gemcitabine and Abraxane), or TGFRt15-TGFRs alone. In addition to treatment cycles, the tumor size of each animal was measured and recorded every other day until termination of the experiment two months after injection of SW1990 cells. Tumor volume measurements were analyzed by group and the results showed that animals receiving the combination of chemotherapy and TGFRt15-TGFRs had significantly smaller tumors compared to the PBS group, whereas chemotherapy was also associated with TGFRt15-TGFRs It was shown that therapy alone also did not work as well as the combination (Figure 107).

In Vitro Senescent B16F10 Melanoma Model In vitro killing of senescent B16F10 melanoma cells by activated mouse NK cells was then assessed. B16F10 senescent (B16F10-SNC) cells were labeled with CellTrace violet and isolated from the spleens of C57BL/6 mice injected with different E:T ratios of in vitro 2t2-activated mouse NK cells (TGFRt15-TGFRs at 10 mg/kg for 4 days). separated) and incubated for 16 hours. Cells were trypsinized, washed and resuspended in complete medium containing propidium iodide (PI) solution. Cytotoxicity was assessed by flow cytometry (Figure 108).

Example 54: Generation and Characterization of 7t15-21s137L (Long Version) Fusion Protein A fusion protein complex consisting of an IL-21/IL-15RαSu/CD137L fusion protein and an IL-7/TF/IL-15 fusion protein was generated (Figures 109 and 110). Specifically, a construct was made by joining the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by the N-terminal coding region of IL-15. The nucleic acid and protein sequences of a construct containing IL-7 attached to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the 7t15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL7)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including the leader sequence) of the 7t15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid and protein sequences for 21s137L are shown below. The nucleic acid sequence (including signal peptide sequence) of the 21s137L construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCAGGCTCCGAGGACTCC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
((G4S)3 linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(human CD137L)
CGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCGCAGCTGGTGGCCCAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGC CTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG GCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGG GACTCTTCCGGGTGACCCCCGAAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including the leader sequence) of the 21s137L fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
((G4S)3 linker)
GGGGSGGGGSGGGGGS
(human CD137L)
REGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSSVSLALHLQPLRSAAGAAALALTVDLLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The IL-21/IL-15RαSu/CD137L and IL-7/TF/IL-15 constructs were cloned into modified retroviral expression vectors 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 vector was transfected into CHO-K1 cells. . Co-expression of these two constructs in CHO-K1 cells results in the formation and secretion of a soluble IL-7/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complex (termed 7t15-21s137L). , which can be purified by anti-TF antibody IgG1 affinity and other chromatographic methods.

Purification Elution Chromatography of 7t15-21s137L Using Anti-TF Antibody Affinity Column 7t15-21s137L harvested from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. As shown in Figure 111, the anti-TF antibody affinity column bound to 7t15-21s137L containing TF as a fusion partner. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step. Figure 112 shows the analytical SEC profile of 7t15-21s137L.

Example 55: Generation and characterization of 7t15-21s137L (short version) fusion protein A fusion protein complex consisting of an IL-21/IL-15RαSu/CD137L fusion protein and an IL-7/TF/IL-15 fusion protein was generated . Specifically, a construct was made by joining the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by the N-terminal coding region of IL-15. The nucleic acid and protein sequences of a construct containing IL-7 attached to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the 7t15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL7)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the 7t15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid and protein sequences for 21s137L (short version) are shown below. The nucleic acid sequence of the 21s137L (short version) construct (including signal peptide sequence) is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
((G4S)3 linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(human CD137 ligand short version)
GATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGG TGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCT CCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGCGCGCCACAGTCTTGGGACTCTTCCGGGTGACCCCCCGAAATC

The amino acid sequence (including signal peptide sequence) of the 21s137L (short version) construct is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
((G4S)3 linker)
GGGGSGGGGSGGGGGS
(human CD137 ligand short version)
DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWQLTQGATVLGLFRVTPEI

The IL-21/IL-15RαSu/CD137L (short version) and IL-7/TF/IL-15 constructs were cloned into modified retroviral expression vectors 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 conveyors highly active T-cell effector functions.Hum Gene Th er 2005;16:457-72), the expression vector was transformed into CHO-K1 cells. was transfected into Co-expression of these two constructs in CHO-K1 cells yielded a soluble IL-7/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complex (termed 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 (4.1BB) On day 1, 96-well plates were coated with 100 μL (2.5 μg/mL) of GAH IgG Fc (G-102) in R5 (coating buffer). -C, R&D Systems) or R5 alone and incubated overnight at 4°C. On day two, plates were washed three times and blocked with 300 μL of 1% BSA in PBS for 2 hours at 37°C. 10 ng/mL 4.1BB/Fc (838-4B, R&D Systems) was added at 100 μL/well and incubated for 2 hours at room temperature. After three washes, 7t15-21s137L or 7t15-21s were serially diluted in a 1/3 ratio (starting at 10 nM) and incubated overnight at 4°C. On day 3, after 3 washes, 300 ng/mL biotinylated anti-hTF antibody (BAF2339, R&D Systems) was added at 100 μL/well and incubated for 2 hours at room temperature. Plates were then washed three times and incubated with 0.25 μg/mL HRP-SA (Jackson ImmuneResearch) for 30 minutes at 100 μL/well, followed by three washes and incubation with 100 μL ABTS for 2 minutes at room temperature. Absorbance was read at 405 nm. As shown in Figure 113, 7t15-21s137L (short version) showed a significant interaction with 4.1BB/Fc (blue line) compared to 7t15-21s.

Detection of IL-15, IL-21 and IL-7 in 7t15-21s137L (short version) using ELISA A 96-well plate was coated with 100 μL (8 μg/mL) anti-TF antibody IgG1 in R5 (coating buffer). Coated and incubated for 2 hours at room temperature. Plates were washed three times and blocked with 100 μL of 1% BSA in PBS. 7t15-21s137L (short version) serially diluted in a 1:3 ratio was added and incubated for 60 minutes at room temperature. After three washes, 50 ng/mL biotinylated anti-IL-15 antibody (BAM247, R&D Systems), 500 ng/mL biotinylated anti-IL21 antibody (13-7218-81, R&D Systems), or 500 ng/mL biotin. Anti-IL7 antibody (506602, R&D Systems) was added to the wells and incubated for 60 minutes at room temperature. Washed 3 times and incubated with 0.25 μg/mL HRP-SA (Jackson ImmunoResearch) at 100 μL/well for 30 minutes at room temperature, then washed 4 times and incubated with 100 μL ABTS for 2 minutes at room temperature. Absorbance was read at 405 nm. As shown in Figures 114A-114C, the IL-15, IL-21, and IL-7 domains in 7t15-21s137L (short version) were detected by the respective antibodies.

IL-15 in 7t15-1s137L (Short Version) Promotes Proliferation of IL2Rαβγ-Containing CTLL2 Cells was compared to recombinant IL-15 for promotion of proliferation of CTLL2 cells expressing IL-15 dependent CTLL2 cells were washed 5 times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serially diluted 7t15-21s137L (short version) or IL-15 were added to the cells (Figure 115). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μL of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 115, 7t15-21s137L (short version) and IL-15 promoted proliferation of CTLL2 cells. The _EC50 for 7t15-21s137L (short version) and IL-15 were 55.91 pM and 6.22 pM, respectively.

IL-21 in 7t15-1s137L (Short Version) Promotes Proliferation of IL21R-Containing B9 Cells To assess the IL-21 activity of 7t15-21s137L (short version) Recombinant IL-21 was compared for promotion of proliferation of B9 cells expressing -21R. IL-21R-containing B9 cells were washed five times with RPMI-10% FBS and seeded in wells at 1×10 ⁴ cells/well. Serially diluted 7t15-21s137L (short version) or IL-21 were added to the cells (Figure 116). Cells were incubated for 5 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μL of WST1 to each well on day 5 and incubating for an additional 4 hours at 37° C. in a CO ₂ incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 116, 7t15-21s137L (short version) and IL-21 promoted proliferation of B9 cells. The EC ₅₀ for 7t15-21s137L (short version) and IL-21 were 104.1 nM and 72.55 nM, respectively.

Example 56 Generation and Characterization of 7t15-TGFRs Fusion Proteins A fusion protein complex consisting of a TGFβ receptor II/IL-15RαSu fusion protein and an IL-7/TF/IL-15 fusion protein was generated (FIG. 117 and FIG. 118). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, IL-15, and IL-7 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a construct was made by joining the IL-7 sequence to the N-terminal coding region of tissue factor 219, followed by the N-terminal coding region of IL-15. The nucleic acid and protein sequences of a construct containing IL-7 attached to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the 7t15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL7)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the 7t15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFSSAYS
(human IL7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by directly coupling two TGFβ receptor II to IL-15RαSu chains synthesized by Genewiz. The nucleic acid and protein sequences of a construct containing TGFβ receptor II attached to the N-terminus of IL-15RαSu are shown below.

The nucleic acid sequences of the TGFRs constructs (including signal peptide sequences) are as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

The amino acid sequence (including leader sequence) of the TGFRs fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFSSAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

Effect of 7t15-TGFRs on TGFβ1 Activity in HEK-Blue TGFβ Cells To assess the activity of TGFβR in 7t15-TGFRs, the effect of 7t15-TGFRs on TGFβ1 activity in HEK-Blue TGFβ cells was analyzed. HEK-Blue TGFβ cells (Invivogen) were washed twice with prewarmed PBS and plated 5× in test medium (DMEM, 10% heat-inactivated FCS, 1× glutamine, 1× anti-anti, and 2× glutamine). Resuspend at 10 ⁵ cells/mL. In a flat bottom 96-well plate, 50 μL of cells were added to each well (2.5×10 ⁴ cells/well) followed by 50 μL of 0.1 nM TGFβ1 (R&D systems). 7t15-TGFRs or TGFR-Fc (R&D Systems) prepared in 1:3 serial dilutions were then added to the plate to a total volume of 200 μL. After 24 hours of incubation at 37°C, 40 μL of induced HEK-Blue TGFβ cell supernatant is added to 160 μL of prewarmed QUANTI-Blue (Invivogen) in a flat bottom 96-well plate and incubated for 1-3 hours at 37°C. bottom. OD values were then determined at 620-655 nM using a plate reader (Multiscan Sky). Data are shown in FIG. The _IC50 for each protein sample was calculated using GraphPad Prism 7.04. The _IC50s for 7t15-TGFRs and TGFR-Fc were 1142 pM and 558.6 pM, respectively. These results indicated that TGFβR in 7t15-TGFRs could block TGFβ1 activity in HEK-Blue TGFβ cells.

Detection of IL-15, TGFβRII, and IL-7 in 7t15-TGFRs Using ELISA A 96-well plate was coated with 100 μL (8 μg/mL) of anti-TF antibody IgG1 in R5 (coating buffer) and incubated at room temperature (RT ) for 2 hours. Plates were washed three times and blocked with 100 μL of 1% BSA in PBS. Serial dilutions of 7t15-TGFRs (1:3 ratio) were added and incubated for 60 minutes at room temperature. After three washes, 50 ng/mL biotinylated anti-IL-15 antibody (BAM247, R&D Systems), 200 ng/mL biotinylated anti-TGFbRII antibody (BAF241, R&D Systems), or 500 ng/mL biotinylated anti-IL-15 antibody (BAF241, R&D Systems). 7 antibody (506602, R&D Systems) was added and incubated for 60 minutes at room temperature. After 3 washes, cells were incubated with 0.25 μg/mL HRP-SA (Jackson ImmunoResearch) at 100 μL/well for 30 minutes at room temperature, followed by 4 washes and incubation with 100 μL ABTS for 2 minutes at room temperature. Absorbance was read at 405 nm. As shown in Figures 120A-120C, IL-15, TGFR, and IL-7 in 7t15-TGFRs were detected by their respective antibodies.

IL-15 in 7t15-TGFRs Promotes Proliferation of 32Dβ Cells Containing IL-2Rβ and Common γ Chain 32Dβ cells expressing the chains were used to compare with recombinant IL-15 to assess their effect on promoting cell proliferation. IL-15 dependent 32Dβ cells were washed 5 times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serially diluted 7t15-TGFRs or IL-15 were added to the cells (Figure 121). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μL of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 121, 7t15-TGFRs and IL-15 promoted 32Dβ cell proliferation and the _EC50 of 7t15-TGFRs and IL-15 were 126 nM and 16.63 pM, respectively.

Purification Elution Chromatography of 7t15-TGFRs Using Anti-TF Antibody Affinity Column 7t15-TGFRs harvested from cell culture were loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. As shown in Figure 122, the anti-TF antibody affinity column can bind 7t15-TGFRs with TF as a fusion partner for 7t15-TGFRs. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Reducing SDS-PAGE Analysis of 7t15-TGFRs To determine protein purity and molecular weight, 7t15-TGFRs protein samples purified with anti-TF antibody affinity columns were run on sodium dodecyl sulfate polyacrylamide gels (4-12%) under reducing conditions. % NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method. After electrophoresis, gels were stained with InstantBlue for approximately 30 minutes and then destained overnight in purified water.

To verify that the 7t15-TGFRs proteins undergo post-translational glycosylation in CHO cells, deglycosylation experiments were performed using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer's instructions. FIG. 123 shows reducing SDS-PAGE analysis of samples in the non-deglycosylated state (lane 1 in red border) and deglycosylated state (lane 2 in yellow border). These results indicated that the protein was glycosylated when expressed in CHO cells. After deglycosylation, purified samples showed the expected molecular weights (55 kDa and 39 kDa) in reducing SDS gels. Lane M was loaded with 10ul of SeeBlue Plus2 Prestained Standard.

Characterization of 7t15-TGFRs 7t15-TGFRs consist of a first polypeptide that is a soluble fusion (7t15) of human IL-7, a human tissue factor 219 fragment, and human IL-15, and two single-chain TGFβRIIs. and a second polypeptide that is a soluble fusion of the sushi domain of the human IL-15 receptor alpha chain (TGFRs) (type A multichain polypeptide as described herein). .

CHO cells were co-transfected with 7t15 and TGFRs vectors. 7t15-TGFRs complexes were purified from transfected CHO cell culture supernatants. IL-7, IL-15, TGFβ receptor, and tissue factor (TF) components in the complex were demonstrated by ELISA, as shown in FIG. Humanized anti-TF monoclonal antibody (anti-TF antibody IgG1) was used as capture antibody to determine TF in 7t15-TGFRs, human IL-15 (R&D systems), human IL-7 (Biolegend), anti-TGFβ Biotinylated antibodies to the receptors (R&D Systems) were used as detection antibodies to determine IL-7, IL-15, and TGFβ receptors in the 7t15-TGFRs, respectively. Bound biotinylated antibody was then detected using peroxidase-conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS substrate (Surmodics IVD, Inc.). Results were analyzed by ELISA (Figure 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 with control solution (PBS) or 0.3, 1, 3, and 10 mg. /kg of 7t15-TGFRs subcutaneously. Treated mice were euthanized. Mouse spleens were harvested and weighed 4 days after treatment. Single splenocyte suspensions were prepared, stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 antibodies, and the percentages of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells were determined by flow cytometry. analyzed. Results showed that 7t15-TGFRs were effective in expanding splenocytes, especially at 1-10 mg/kg, based on spleen weight (FIG. 125A). The percentages of CD8 ⁺ T cells and NK cells were higher compared to control treated mice at all doses tested (Fig. 125B).

CD44 Expression on CD4+ and CD8+ T Cells IL-15 is known to induce CD44 expression on T cells and the growth of memory T cells. CD44 expression on CD4 ⁺ and CD8 ⁺ T cells in mice treated with 7t15-TGFRs was assessed. C57BL/6 mice were treated subcutaneously with 7t15-TGFRs. Splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-CD44 monoclonal antibodies for immune cell subsets. The percentage of CD4 ⁺ CD44 ^high T cells of total CD4 ⁺ T cells and CD8 ⁺ CD44 ^high T cells of total CD8 ⁺ T cells was analyzed by flow cytometry. As shown in Figures 126A and 126B, 7t15-TGFRs significantly activated CD4 ⁺ and CD8 ⁺ T cells to differentiate into memory T cells.

In addition, dynamic proliferation of immune cells based on splenocyte Ki67 expression and cytotoxic potential based on splenocyte granzyme B expression induced by 7t15-TGFRs after single-dose treatment of mice were also evaluated. C57BL/6 mice were treated subcutaneously with 3 mg/kg 7t15-TGFRs. Treated mice were euthanized and splenocytes were prepared. Prepared splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies for immune cell subsets, followed by anti-Ki67 antibody for cell proliferation and anti-Ki67 antibody for cytotoxicity markers. Intracellular staining was performed with a granzyme B antibody. Mean fluorescence intensity (MFI) of Ki67 and granzyme B of corresponding immune cell subsets was analyzed by flow cytometry. As shown in Figures 127A and 127B, the expression of Ki67 and granzyme B by CD8 ⁺ T cells and NK cells was increased in the spleens of mice treated with 7t15-TGFRs compared to PBS control treatment. These results indicate that 7t15-TGFRs not only increase the numbers of CD8 ⁺ T cells and NK cells, but also enhance the cytotoxic potential of these cells.

In addition, the cytotoxicity of mouse splenocytes against tumor cells was also evaluated. Mouse Yac-1 cells were labeled with CellTrace Violet and used as tumor target cells. Splenocytes were prepared from mice treated with 7t15-TGFRs and used as effector cells. Target cells were mixed with effector cells at an E:T ratio of 10:1 in RPMI-10 medium with or without 100 nM 7t15-TGFRs and incubated at 37° C. for 20 hours. Targeted Yac-1 cell inhibition was assessed by analysis of viable violet-labeled Yac-1 cells using flow cytometry. The percentage of Yac-1 inhibition was calculated using the formula (1−number of viable Yac-1 cells in experimental sample/number of viable Yac-1 cells in sample without splenocytes)×100. As shown in Figure 128, mouse splenocytes treated with 7t15-TGFRs exhibited stronger cytotoxicity against Yac-1 cells than control mouse splenocytes, indicating that 7t15-TGFRs were added during the cytotoxicity assay. This further enhanced splenocyte cytotoxicity against Yac-1 target cells.

Example 57: Generation and Characterization of TGFRt15-21s137L Fusion Protein A fusion protein complex comprising an IL-21/IL-15RαSu/CD137L fusion protein and a TGFβ receptor II/TF/IL-15 fusion protein was generated (FIG. 129). and FIG. 130). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a G4S(3) linker was used to join two TGFβ receptor II sequences to generate a single-chain version of TGFβ receptor II, followed by the N-terminal coding region of tissue factor 219 followed by A construct was made by directly ligating to the N-terminal coding region of IL-15.

The nucleic acid sequence (including signal peptide sequence) of the TGFRt15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the TGFRt15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid and protein sequences for 21s137L are shown below. The nucleic acid sequence (including signal peptide sequence) of the 21s137L construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCAGGCTCCGAGGACTCC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
((G4S)3 linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(human CD137L)
CGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCGCAGCTGGTGGCCCAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGC CTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG GCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGG GACTCTTCCGGGTGACCCCCGAAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including the leader sequence) of the 21s137L fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
((G4S)3 linker)
GGGGSGGGGSGGGGGS
(human CD137L)
REGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSSVSLALHLQPLRSAAGAAALALTVDLLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The IL-21/IL-15RαSu/CD137L and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors previously described (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. 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 vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allows formation and secretion of a soluble TGFR/TF/IL-15:IL-21/IL-15RαSu/CD137L protein complex (termed TGFRt15-21s137L). , 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 harvested from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. As shown in Figure 131, the anti-TF antibody affinity column bound to TGFRt15-21s137L containing TF as a fusion partner of TGFRt15-21s137L. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Example 58: Generation and Characterization of TGFRt15-TGFRs21 Fusion Proteins A fusion protein complex consisting of a TGFβ receptor II/IL-15RαSu/IL-21 fusion protein and a TGFβ receptor II/TF/IL-15 fusion protein was generated (Figures 132 and 133). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, IL-21, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a G4S(3) linker was used to join two TGFβ receptor II sequences to generate a single-chain version of TGFβ receptor II, followed by the N-terminal coding region of tissue factor 219 followed by A construct was made by directly ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of a construct containing two TGFβ receptor II bound to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the TGFRt15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the TGFRt15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by linking two TGFβ receptor II directly to the IL-15RαSu chain synthesized by Genewiz, followed by the N-terminal coding region of IL-21. The nucleic acid and protein sequences of a construct containing TGFβ receptor II attached to the N-terminus of IL-15RαSu followed by the N-terminus of IL-21 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the TGFRs21 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCAGGCTCCGAGGACTCC

The amino acid sequence (including leader sequence) of the TGFRs21 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The TGFR/IL-15RαSu/IL-21 and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors previously described (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. 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 vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allowed the formation and secretion of a soluble TGFR/TF/IL-15:TGFR/IL-15RαSu/IL-21 protein complex (termed TGFRt15-TGFRs21). , 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 collected from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 KDa. As shown in Figure 134, the anti-TF antibody affinity column bound TGFRt15-TGFRs21 with TF as a fusion partner. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Reducing SDS-PAGE Analysis of TGFRt15-TGFRs21 To determine protein purity and molecular weight, anti-TF antibody affinity column-purified TGFRt15-TGFRs21 protein samples were run on sodium dodecyl sulfate polyacrylamide gels (4-12%) under reducing conditions. % NuPage Bis-Tris gel) electrophoresis (SDS-PAGE) method. After electrophoresis, gels were stained with InstantBlue for approximately 30 minutes and then destained overnight in purified water.

To verify that the TGFRt15-TGFRs21 proteins undergo post-translational glycosylation in CHO cells, deglycosylation experiments were performed using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer's instructions. FIG. 135 shows reducing SDS-PAGE analysis of samples in the non-deglycosylated state (lane 1 in red border) and deglycosylated state (lane 2 in yellow border). It is clear that the protein is glycosylated when expressed in CHO cells. After deglycosylation, purified samples showed the expected molecular weights (69 kDa and 55 kDa) in reducing SDS gels. Lane M was loaded with 10ul of SeeBlue Plus2 Prestained Standard.

Immunostimulation of TGFRt15-TGFRs21 in C57BL/6 Mice TGFRt15-TGFRs21 is a first polypeptide that is a single-chain soluble fusion of two TGFβRII domains, a human tissue factor 219 fragment, and human IL-15 (TGFRt15). and a single-chain two TGFβRII domains, a sushi domain of the human IL-15 receptor alpha chain, and a second polypeptide that is a soluble fusion of human IL-21 (TGFRs21) ( Type A multichain polypeptides described herein).

CHO cells were co-transfected with TGFRt15 and TGFRs21 vectors. TGFRt15-TGFRs21 complexes were purified from transfected CHO cell culture supernatants. As shown in Figure 136, TGFβ receptor, IL-15, IL-21, and tissue factor (TF) components in the complex were demonstrated by ELISA. Using humanized anti-TF monoclonal antibody (anti-TF IgG1) as capture antibody, TF was determined and biotinylated anti-human IL-21 antibody (R&D Systems) was used as detection antibody to determine IL-15, TGFβ receptor and IL-21 in TGFRt15-TGFRs21, respectively. For detection, peroxidase-conjugated streptavidin (Jackson ImmunoResearch Lab) and ABTS were used.

Wild-type C57BL/6 mice were treated subcutaneously with either control solution (PBS) or 3 mg/kg TGFRt15-TGFRs21. After 4 days of treatment, spleen weight and percentage of various immune cell types present in the spleen were assessed. The percentages of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells present in the spleens of control-treated and TGFRt15-TGFRs21-treated mice were assessed, as shown in FIG. 137A. Dynamic proliferation of immune cells based on Ki67 expression after TGFRt15-TGFRs21 treatment was also assessed. Splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies and intracellularly with anti-Ki67 antibody. The percentages of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells and the mean fluorescence intensity (MFI) of Ki67 of the corresponding immune cell subsets were analyzed by flow cytometry (FIGS. 137A and 137B). In addition, the cytotoxic potential based on splenocyte granzyme B expression induced by TGFRt15-TGFRs21 after single-dose treatment of mice was also assessed. As shown in Figure 138, in the spleen of mice treated with TGFRt15-TGFRs21, expression of granzyme B by NK cells increased after treatment. Splenocytes from TGFRt15-TGFRs21 treated mice were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies and intracellularly with anti-granzyme B antibody. Granzyme B mean fluorescence intensity (MFI) of the corresponding immune cell subsets was analyzed by flow cytometry (Figure 138).

As shown in FIG. 137A, in the spleen of mice treated with TGFRt15-TGFRs21, the percentage of both CD8 ⁺ T cells and NK cells increased 4 days after single TGFRt15-TGFRs21 treatment. These results indicate that TGFRt15-TGFRs21 can induce immune cells to proliferate mouse spleens, especially CD8 ⁺ T cells and NK cells.

In addition, the cytotoxicity of mouse splenocytes against tumor cells was also evaluated. Mouse Yac-1 cells were labeled with CellTrace Violet and used as tumor target cells. Splenocytes were prepared from mice treated with TGFRt15-TGFRs21 and used as effector cells. Target cells were mixed with effector cells at an E:T ratio of 10:1 in RPMI-10 medium with or without 100 nM TGFRt15-TGFRs21 and incubated at 37° C. for 24 hours. Targeted Yac-1 cell inhibition was assessed by analysis of viable violet-labeled Yac-1 cells using flow cytometry. The percentage of Yac-1 inhibition was calculated using the formula (1−[number of viable Yac-1 cells in experimental sample]/[number of viable Yac-1 cells in sample without splenocytes])×100. . As shown in Figure 139, mouse splenocytes treated with TGFRt15-TGFRs21 exhibited stronger cytotoxicity against Yac-1 cells than control mouse cells in the presence of TGFRt15-TGFRs21 during the cytotoxicity assay. (Fig. 139).

Example 59 Generation of TGFRt15-TGFRs16 Fusion Proteins A fusion protein complex consisting of a TGFβ receptor II/IL-15RαSu/anti-CD16 scFv fusion protein and a TGFβ receptor II/TF/IL-15 fusion protein was generated (FIGS. 140 and Figure 141). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a G4S(3) linker was used to join two TGFβ receptor II sequences to generate a single-chain version of TGFβ receptor II, followed by the N-terminal coding region of tissue factor 219 followed by A construct was made by directly ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of a construct containing two TGFβ receptor II bound to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the TGFRt15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the TGFRt15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by linking two TGFβ receptor II directly to IL-15RαSu chains synthesized by Genewiz followed by anti-CD16 scFv sequences. The nucleic acid and protein sequences of a construct containing TGFβ receptor II linked to the N-terminus of IL-15RαSu followed by an anti-CD16 scFv sequence are shown below.

The nucleic acid sequence (including signal peptide sequence) of the TGFRs16 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
(anti-human CD16scFv)
TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACACAGGCCCTCCGGCATCCCTGACAGG TTCTCCGGATCCCTCCTCCGGCAACACCGCCTCCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCGGCAACCATGTGGGTGTTCGGCGGCGGGCACCAAGCTGACCGTGGGCCATGGCGGCGGCGGCTGTCCGGAGGCGGCCG GCAGCGGCGGAGGAGGATCCGAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAGGGGCCTGGAGTGGGTGT CCGGCATCAACTGGAACGGCGGATCCACCGGCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGA CAGGGCACCCTGGTGACCGTGTCCCAGG

The amino acid sequence (including leader sequence) of the TGFRs16 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
(anti-human CD16scFv)
SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGHGGGGGSGGGGSGGGGSEVQLVESGGGGVVRPGG SLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The TGFR/IL-15RαSu/anti-CD16 scFv and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors 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;16:4 57-72), the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allowed formation and secretion of a soluble TGFR/TF/IL-15:TGFR/IL-15RαSu/anti-CD16scFv protein complex (termed TGFRt15-TGFRs16). , which can be purified by anti-TF IgG1 affinity and other chromatographic methods.

Example 60 Generation of TGFRt15-TGFRs137L Fusion Protein A fusion protein complex consisting of a TGFβ receptor II/IL-15RαSu/CD137L fusion protein and a TGFβ receptor II/TF/IL-15 fusion protein was generated (FIGS. 142 and 142). 143). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, CD137L, and IL-15 sequences were obtained from the UniProt website and DNA for these sequences was synthesized by Genewiz. Specifically, a G4S(3) linker was used to join two TGFβ receptor II sequences to generate a single-chain version of TGFβ receptor II, followed by the N-terminal coding region of tissue factor 219 followed by A construct was made by directly ligating to the N-terminal coding region of IL-15.

The nucleic acid and protein sequences of a construct containing two TGFβ receptor II bound to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

The nucleic acid sequence (including signal peptide sequence) of the TGFRt15 construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the TGFRt15 fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Constructs were also made by linking two TGFβ receptor II directly to the IL-15RαSu chain synthesized by Genewiz, followed by the (G4S)3 linker and CD137L sequence. The nucleic acid and protein sequences of a construct containing TGFβ receptor II attached 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 signal peptide sequence) of the TGFRs137L construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(Human TGFβ receptor II fragment)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
((G4S)3 linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(human CD137L)
CGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCGCAGCTGGTGGCCCAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGC CTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG GCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGG GACTCTTCCGGGTGACCCCCGAAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including the leader sequence) of the TGFRs137L fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(human TGFβ receptor II)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
((G4S)3 linker)
GGGGSGGGGSGGGGGS
(human CD137L)
REGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSSVSLALHLQPLRSAAGAAALALTVDLLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSSE

In some cases, the leader peptide is cleaved from the intact polypeptide to generate the mature form, which can be soluble or secreted.

The TGFR/IL-15RαSu/CD137L and TGFR/TF/IL-15 constructs were cloned into modified retroviral expression vectors 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;16:4 57-72), the expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allowed the formation and secretion of a soluble TGFR/TF/IL-15:TGFR/IL-15RαSu/CD137L protein complex (termed TGFRt15-TGFRs137L). , which can be purified by anti-TF IgG1 affinity and other chromatographic methods.

Example 61. Production and Characterization of an Exemplary Single Chain Chimeric Polypeptide 2t2 A First Target Binding Domain That Binds the IL-2 Receptor, a Soluble Human Tissue Factor Domain, and a Second Target Binding that Binds the IL-2 Receptor An exemplary single-chain chimeric polypeptide containing the domain was generated (IL-2/TF/IL-2, designated 2t2) (Figure 144). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.
Nucleic Acid (SEQ ID NO: 164) Encoding an Exemplary Single Chain Chimeric Polypeptide (IL-2/TF/IL-2)
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(First IL-2 fragment)
GCCCCCACCTCCTCCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCCAAGCTGACTCGTATGCTGACCTTCAAGTTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGA GCTGAAGCCCCTCGAGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAATCAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGG ATCACCTTCTGCCAGTTCCATCATCTCCACTTTAACC
(human tissue factor 219 form)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(second IL-2 fragment)
GCACCTACTTCAAGTTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCCACCAGGATGCTCACATTTTAAGTTTTTACATGCCCAGAAGGCCCACAGAACTGAACATCTTCAGTGTCTAGAAGA AGAACTCAAAACCTCTGGAGGAAGTGCTAAAATTTAGCTACAAAGCAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTTCTGGAACTAAAGGGATCTGAAAACAATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCT TTTGTCAAAGCATCATCTCTCAACACTAACT
Exemplary Single Chain Chimeric Polypeptide (IL-2/TF/IL-2) (SEQ ID NO: 163)
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-2)
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-2)
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

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 allowed secretion of a soluble IL-2/TF/IL-2 single-chain chimeric polypeptide (designated 2t2), which was analyzed for anti-TF antibody affinity and other chromatographic It can be purified by graphic methods.

IL-2 and 2t2 promoted IL-2Rβ and common γ chain-containing 32Dβ cell proliferation in a similar manner. 2t2 was compared to recombinant IL-2 for promotion of proliferation. IL-2-dependent 32Dβ cells were washed five times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serial dilutions of 2t2 or IL-2 were added to the cells (Figure 145). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μl of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ 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 32Dβ cells in a similar manner. The _EC50 for 2t2 and IL-2 were 158.1 pM and 140 pM, respectively.

2t2 showed an improved ability to promote IL-2Rαβγ, including CTLL-2 cell proliferation compared to IL-2 To assess the IL-2 activity of 2t2, IL-2Rα, IL-2Rβ , and promotion of proliferation of CTLL-2 cells expressing the common γ chain, 2t2 was compared to recombinant IL-2. IL-2 dependent CTLL-2 cells were washed 5 times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serial dilutions of 2t2 or IL-2 were added to the cells (Figure 146). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μl of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 146, 2t2 promoted proliferation of CTLL-2 cells 4- to 5-fold more potent than IL-2. The _EC50 for 2t2 was 123.2 pM and IL-2 was 548.2 pM.

2t2 suppressed hyperfat-induced increases in hyperglycemia in ApoE ^{−/ −} mice They were fed a high-fat diet (TD88137, Harlan Laboratories) containing % cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch and maintained under standard conditions. At week 7, mice fed the high-fat diet were randomly assigned to control and treatment groups. Mice then received either 2t2 per subcutaneous injection (treatment group) or PBS (diet and control groups) at a dosage of 3 mg/kg. Three days after dosing, mice were fasted overnight and blood samples were collected by retro-orbital venous puncture. Overnight fasting glucose levels were measured using a OneTouch blood glucose meter. As shown in FIG. 147, the results showed that 2t2 injection effectively suppressed elevated glucose levels in ApoE ^−/− mice.

2t2 significantly upregulates the proportion of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory (Treg) cells in blood lymphocytes Six-week-old female ApoE ^−/− mice (Jackson Lab) were fed a standard chow diet. , or a high-fat diet containing 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch (TD88137, Harlan Laboratories) and maintained under standard conditions. At week 7, mice fed the high-fat diet were randomly assigned to control and treatment groups. Mice then received either 2t2 per subcutaneous injection (treatment group) or PBS (diet and control groups) at a dosage of 3 mg/kg. Three days after dosing, overnight fasting blood samples were collected by retro-orbital venous puncture and incubated with ACK lysis buffer (Thermo Fisher Scientific) for 5 minutes at 37°C. Samples were then resuspended in FACS buffer (1X PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and FITC-anti-CD4 and APC- Surface stained with anti-CD25 antibody (BioLegend) for 30 minutes. Surface-stained samples were further fixed, premetallized with Fix/Perm buffer (BioLegend) and intracellularly stained with PE-anti-Foxp3 antibody (BioLegend). After staining, cells were washed twice with FACs buffer and then centrifuged at 1500 RPM for 5 minutes at room temperature. Cells were analyzed by flow cytometry (Celesta-BD Bioscience). As shown in Figure 148, 2t2 treatment increased blood It significantly increased the Treg population of lymphocytes (3.5%±0.32).

Purification Elution Chromatography of 2t2 from Anti-TF Antibody Affinity Column 2t2 harvested from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 kDa. As shown in Figure 149, the anti-TF antibody affinity column bound 2t2 containing TF as the fusion domain. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Analytical Size Exclusion Chromatography (SEC) Analysis of 2t2 To analyze 2t2 using analytical size exclusion chromatography (SEC), a Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was run on AKTA Avant. It was connected to a system (manufactured by GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. Samples containing 2t2 in PBS were injected onto a Superdex 200 column using a capillary loop and analyzed by SEC. An SEC chromatograph of the sample is shown in FIG. SEC results showed two protein peaks of 2t2.

Reducing SDS-PAGE of 2t2
To determine protein purity and molecular weight, anti-TF antibody affinity column purified 2t2 protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS- PAGE) method. After electrophoresis, gels were stained with InstantBlue for approximately 30 minutes and then destained overnight in purified water.

To verify that the 2t2 protein undergoes post-translational glycosylation in CHO cells, deglycosylation experiments were performed using the Protein Deglycosylation Mix II kit from New England Biolabs according to the manufacturer's instructions. Figures 151A and 151B show reducing SDS-PAGE analysis of samples in the non-deglycosylated state (lane 1 in red border) and deglycosylated state (lane 2 in yellow border). Results indicated that the 2t2 protein was glycosylated when expressed in CHO cells. After deglycosylation, purified samples were run at the expected molecular weight (56 kDa) in reducing SDS gels. Lane M was loaded with 10 μL of SeeBlue Plus2 Prestained Standard.

In Vivo Characterization of 2t2 2t2 was injected subcutaneously into C57BL/6 mice at various doses to determine the immunostimulatory activity of 2t2 in vivo. Mice were treated subcutaneously with control solution (PBS) or 0.1, 0.4, 2, and 10 mg/kg of 2t2. Treated mice were euthanized 3 days after treatment. Spleens of mice were harvested and weighed 3 days after treatment. A single splenocyte suspension was prepared and the prepared splenocytes were stained for CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells (with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-NK1.1 antibodies). , analyzed by flow cytometry. The results showed that 2t2 was effective in expanding splenocytes, especially at 0.1-10 mg/kg, based on spleen weight (Figure 152A). The percentage of CD8 ⁺ T cells was higher compared to control treated mice at 2 and 10 mg/kg (Fig. 152B). The percentage of NK cells was higher compared to control-treated mice at all doses tested (Fig. 152B).

IL-2 is known to upregulate CD25 expression by immune cells. Therefore, CD25 expression on CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells in 2t2-treated mice was accessed. C57BL/6 mice were treated subcutaneously with 2t2 as described in the paragraph above. Splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, CD25, and NK1.1 monoclonal antibodies. CD25 expression (MFI) of splenocyte subsets was analyzed by flow cytometry. As shown in Figure 153, at the doses and time points tested, 2t2 significantly upregulated CD25 expression by CD4 ⁺ T cells, but not CD8 ⁺ T cells or NK cells.

The pharmacokinetics of 2t2 in C57BL/6 mice was also investigated. 2t2 was injected subcutaneously into C57BL/6 mice at 1 mg/kg. Mice were bled from the tail vein at various time points and sera were prepared, as shown in FIG. 2t2 concentrations were measured using an ELISA (capture: anti-tissue factor antibody; detection: biotinylated anti-human IL-2 antibody followed by SA-HRP and ABTS substrate). The half-life of 2t2 was 1.83 hours calculated using PK Solutions 2.0 (Summit Research Services).

2t2 Attenuated High Fat-Induced Atherosclerotic Plaque Formation in ApoE ^{−/ −} Mice (21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% starch) (TD88137, Harlan Laboratories) and maintained under standard conditions. At week 7, mice fed a high-fat diet (HFD) were randomly assigned to control and treatment groups. Mice were then dosed subcutaneously weekly for 4 weeks with either 2t2 (treatment group) or PBS (solid food group and control group) at a dosage of 3 mg/kg. At 12 weeks, all mice were euthanized with isoflurane. Aortas were harvested, opened longitudinally and stained with Sudan IV solution (0.5%) using the en face method. The percentage of plaque area (red, as shown in Figure 155A) relative to total aortic area was then quantified with ImageJ software. FIG. 155A shows representative views of atherosclerotic plaques from each group. FIG. 155B shows the results of quantitative analysis of atherosclerotic plaques in each group. The percentage of plaque area in the control group (HF diet) was much higher than in the treatment group (HFD+2t2), 10.28% vs. 4.68%.

2t2 suppresses type 2 diabetes progression Male BKS. Cg-Dock7 ^m +/+Lepr ^db /J (db/db (Jackson Lab)) mice were fed a standard chow diet and had access to drinking water ad libitum. At 6 weeks of age, mice were randomly assigned to control and treatment groups. The treatment group received 2t2 by subcutaneous injection at 3 mg/kg every other week, while the control group received vehicle only (PBS). Overnight fasting glucose levels were measured weekly using the OneTouch blood glucose meter. The results show that 2t2 is BKS. Cg-Dock7 ^m +/+Lepr ^db /J mice were shown to effectively suppress elevated glucose levels (FIG. 156).

2t2 significantly upregulates the proportion of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cells in blood lymphocytes after the first injection Male BKS. Cg-Dock7 ^m +/+Lepr ^db /J(db/db) (Jackson Laboratory) mice were fed a standard chow diet and had access to drinking water ad libitum. At 6 weeks of age, mice were randomly assigned to control and treatment groups. The treatment group received 2t2 by subcutaneous injection at 3 mg/kg every other week, while the control group received vehicle only (PBS). Four days after the first drug injection, overnight fasting blood samples were collected and incubated with ACK lysis buffer (Thermo Fisher Scientific) for 5 minutes at 37°C. Samples were then resuspended in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) and FITC-anti-CD4 and APC were - Surface staining with anti-CD25 antibody (BioLegend) for 30 minutes. Surface-stained samples were further fixed, premetallized with Fix/Perm buffer (BioLegend) and intracellularly stained with PE-anti-Fox p3 antibody (BioLegend). After staining, cells were washed twice with FACs buffer and analyzed by flow cytometry (Celesta-BD Bioscience). The percentage of CD4 ⁺ CD25 ⁺ FoxP3 ⁺ Tregs in blood lymphocytes was measured. As shown in Figure 157, the results showed that 2t2 significantly upregulated the proportion of Tregs in blood lymphocytes (*p<0.05).

Example 62. Production and Characterization of an Exemplary Single Chain Chimeric Polypeptide 15t15 A First Target Binding Domain That Binds the IL-15 Receptor, a Soluble Human Tissue Factor Domain, and a Second Target Binding that Binds the IL-15 Receptor A second exemplary single-chain chimeric polypeptide containing the domain was generated (IL-15/TF/IL-15, designated 15t15) (Figure 158). The nucleic acid and amino acid sequences of this single-chain chimeric polypeptide are shown below.
Nucleic Acid (SEQ ID NO: 170) Encoding an Exemplary Single Chain Chimeric Polypeptide (IL-15/TF/IL-15)
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(First IL-15 fragment)
AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCCACTCTGTACACTGAGAGCGACGTGCACCCTAGGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGCGATGCCAG CATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTTAGC
(human tissue factor 219 form)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(second IL-15 fragment)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC
Exemplary Single Chain Chimeric Polypeptide (IL-15/TF/IL-15) (SEQ ID NO: 169)
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

Nucleic acids encoding IL-15/TF/IL-15 were cloned into modified retroviral expression vectors as previously described (Hughes et al., Hum Gene Ther 16:457-72, 2005). Expression vectors encoding IL-15/TF/IL-15 were transfected into CHO-K1 cells. Expression of the expression vector in CHO-K1 cells allowed secretion of a soluble IL-15/TF/IL-15 single-chain chimeric polypeptide (designated 15t15), which was tested for anti-TF antibody affinity and other It can be purified by chromatographic methods.

15t15 Promotes Proliferation of IL-2Rβ and Common γ Chain Containing 32Dβ Cells The IL-15 activity of 15t15 was compared to recombinant IL-15 in IL2Rβ and common γ chain expressing 32Dβ cells. IL-15 dependent 32Dβ cells were washed 5 times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serial dilutions of 15t15 or IL-15 were added to the cells (Figure 159). Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 10 μl of WST1 to each well on day 3 and incubating for an additional 3 hours at 37° C. in a CO ₂ incubator. The amount of formazan dye produced was analyzed by measuring the absorbance at 450 nm. As shown in Figure 159, 15t15 did not effectively promote proliferation of 32Dβ cells compared to IL-15. The _EC50 for 15t15 and IL-15 were 161.4 pM and 1.6 pM, respectively.

Purification Elution Chromatography of 15t15 from Anti-TF Antibody Affinity Column 15t15 harvested from cell culture was loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After sample loading, the column was washed with 5 column volumes of PBS and then eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized to pH 7.5-8.0 with 1M Tris base. Neutralized samples were then buffer exchanged into PBS using Amicon centrifugal filters with a molecular weight cutoff of 30 kDa. As shown in Figure 160, the anti-TF antibody affinity column bound to 15t15 containing TF as the fusion domain. Buffer-exchanged protein samples were stored at 2-8° C. for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). The column was then neutralized using 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate was 4 mL/min for all steps except the 2 mL/min elution step.

Reducing SDS-PAGE of 15t15
To determine protein purity and molecular weight, anti-TF antibody affinity column purified 15t15 protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) electrophoresis (SDS- PAGE) method. After electrophoresis, gels were stained with InstantBlue for approximately 30 minutes and then destained overnight in purified water.

To verify that the 15t15 protein undergoes post-translational glycosylation in CHO cells, deglycosylation experiments were performed using the Protein Deglycosylation Mix II kit from New England Biolabs and the manufacturer's instructions. Figures 161A and 161B show reducing SDS-PAGE analysis of samples in the non-deglycosylated state (lane 1 in red border) and deglycosylated state (lane 2 in yellow border). Results indicated that the 15t15 protein was glycosylated when expressed in CHO cells. After deglycosylation, purified samples were run at the expected molecular weight (50 kDa) in reducing SDS gels. Lane M was loaded with 10 μL of SeeBlue Plus2 Prestained Standard.

Example 63: In Vitro Stimulation of NK Cells A series of experiments were performed to assess changes in the surface phenotype of NK cells following stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s plus anti-TF antibodies. . For these experiments, fresh human leukocytes were obtained from a blood bank and CD56 ⁺ NK cells were isolated with RosetteSep/human NK cell reagent (StemCell Technologies). NK cell purity was greater than 90% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclon e) resuspended at 0.2×10 ⁶ /mL in a 96-well flat-bottom plate in Cells were treated with 18t15-12s (100 nM), 18t15-12s16 (100 nM), the single cytokines rhIL-15 (50 ng/mL) (Miltenyi) and rhIL18 (50 ng/mL) (Invivogen) and rhIL-12 (10 ng/mL). (Peprotech), 7t15-21s plus anti-TF antibody (100 nM-50 nM), 7t15-21s (100 nM), or anti-TF antibody (50 nM) for 16 h at 37° C. and 5% CO ₂ . The next day, cells were harvested and surface stained with antibodies specific for CD56, CD16, CD25, CD69, CD27, CD62L, NKp30, and NKp44 for 30 minutes. After surface staining, cells were washed (1500 RPM at room temperature) in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). 5 minutes). After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figures 162A and 162B show the percentage of cells expressing CD25, CD69, NKp44, and NKp30 activation markers by overnight incubation of purified NK cells with 18t15-12s, 18t15-12s16, or 7t15-21s plus anti-TF antibody. increased and decreased the percentage of cells expressing CD62L. All activation marker data are from CD56 ⁺ gated lymphocytes.

A series of experiments were performed to assess changes in the surface phenotype of lymphocyte populations following stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s. For these experiments, fresh human leukocytes were obtained from a blood bank. Peripheral blood lymphocytes were isolated on Ficoll-PAQUE Plus (GE Healthcare) density gradient medium. Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclon e) resuspended at 0.2×10 ⁶ /mL in a 96-well flat-bottom plate in Cells were treated with 18t15-12s (100 nM), 18t15-12s16 (100 nM), the single cytokines rhIL-15 (50 ng/mL) (Miltenyi) and rhIL18 (50 ng/mL) (Invivogen) and rhIL-12 (10 ng/mL). (Peprotech), 7t15-21s (100 nM) plus anti-TF antibody (50 nM), 7t15-21s (100 nM), or anti-TF antibody (50 nM) for 16 h at 37° C. and 5% CO ₂ . The next day, cells were harvested and surface stained with antibodies specific for CD4 or CD8, CD62L, and CD69 for 30 minutes. After surface staining, cells were washed (1500 RPM at room temperature) in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). 5 minutes). After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 163 shows the percentage of CD8 and CD4 T cells expressing CD69 by overnight incubation of purified lymphocyte populations (CD4 and CD8 T cells) with 18t15-12s, 18t15-12s16, or 7t15-21s plus anti-TF antibody. increased. Furthermore, incubation with 7t15-21s plus anti-TF antibody increased the percentage of CD8 and CD4 T cells expressing CD62L (FIG. 163).

A series 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 intracellular biochemical conversion of one molecule of glucose into two molecules of pyruvate with the simultaneous production of two molecules of ATP. Increased glycolysis was indicated by an increase in ECAR measured by a Seahorse XF96 analyzer. For these experiments, fresh human leukocytes were obtained from a blood bank and CD56 ⁺ NK cells were isolated with RosetteSep/human NK cell reagent (StemCell Technologies). NK cell purity was greater than 70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). Cells were counted and added to 0.2 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone ) resuspended at 0.2×10 ⁶ /mL in 96-well flat-bottom plates in medium (supplemented). Cells were treated with a mixture of hIL-12 (10 ng/mL) (Biolegend), hIL-18 (50 ng/mL) (R&D), and hIL-15 (50 ng/mL) (NCI), or 18t15-12s (100 nM). Either was stimulated at 37° C. and 5% CO ₂ for 14-18 hours. The next day, cells were harvested and washed twice with Seahorse medium. Cells (2×10 ⁵ cells/well) were seeded in 96-well flux plates coated with 10 μL of poly-L-lysine (Sigma). NK cells were allowed to adhere to the plates for 30 minutes prior to assay. Glucose, oligomycin, and 2DG solutions were prepared in buffered Seahorse medium at 10x concentrations 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 the average reading over 80 minutes or the average reading at each time point. FIG. 164 shows that overnight stimulation of NK cells with 18t15-12s increased basal ECAR levels. Addition of glucose and oligomycin showed enhanced glycolytic and glycolytic capacity of NK cells stimulated overnight with 18t15-12s, respectively (FIG. 164). NK cells treated overnight with medium alone or a mixture of IL12, IL18, and IL-15 were used for comparison (Figure 164).

A series of experiments were performed to determine the increase in phospho-STAT4 and phospho-STAT5 levels in NK cells after stimulation with 18t15-12s. For these experiments, fresh human leukocytes were obtained from a blood bank and CD56 ⁺ NK cells were isolated with RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 specific antibodies (BioLegend). Cells were counted and added to 0.1 mL complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone ) resuspended at 0.05×10 ⁶ /mL in 96-well flat-bottom plates in medium (supplemented). Cells were incubated with hIL-12 (10 ng/mL) (Biolegend) or hIL-15 (50 ng/mL) (NCI) (single cytokines) or 18t15-12s (100 nM) for 90 minutes at 37° C. and 5% CO ₂ . Stimulated for minutes. Unstimulated NK cells (US) were used as controls. Cells were harvested and fixed with paraformaldehyde (Sigma) to a final concentration of 1.6%. Plates were incubated in the dark for 10 minutes at room temperature. FACS buffer (1×PBS (Hyclone) with 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) was added (100 μL) and the cells plated in 96-well “V” bottom plates. moved. Cells were washed at 1500 RPM for 5 minutes at room temperature. The cell pellet was mixed with 100 μL of chilled methanol by gently pipetting up and down and the cells were incubated at 4° C. for 30 minutes. Cells were mixed with 100 mL FACS buffer and washed at 1500 RPM for 5 minutes at room temperature. Cell pellets were mixed with 4 mL of pSTAT4 (BD Bioscience) and pSTAT5 antibodies (BD Bioscience) in 50 mL of FACS buffer, then incubated for 30 minutes at room temperature in the dark. Cells were mixed with 100 mL FACS buffer and washed at 1500 RPM for 5 minutes at room temperature. Cell pellets were mixed with 50 mL of FACS buffer and cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 165 shows that incubation of NK cells with 18t15-12s induced an increase in pSTAT4 and pSTAT5 (plotted data, normalized fold change).

A series of experiments were performed to demonstrate the effects of 18t15-12s or a mixture of cytokines (eg, IL12, IL18, and IL-15) on the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of NK cells purified from human blood. determined the effect. OCR and ECAR were measured by a Seahorse XF96 analyzer. In these experiments, human NK cells were freshly isolated from human leukemia by negative selection using RosetteSep/human NK cell reagent (StemCell Technologies). Freshly purified NK cells were treated overnight with either 18t15-12s (100 nM) or a mixture of rhIL12 (10 ng/mL), rhIL18 (50 ng/mL), and rhIL-15 (50 ng/mL) cytokines as a control ( 16 hours). The next day, cells were washed, counted, and equal numbers of cells were plated in buffered Seahorse medium. Glucose, oligomycin, and 2DG solutions were prepared in buffered Seahorse medium at 10x concentrations and injected into ports A, B, and C of the calibration plate. Figure 166 shows OCR (left) and ECAR (right) data from two independent donors. 18 shows that overnight stimulation of NK cells with 18t15-12s increased basal ECAR and OCR levels. Addition of glucose and oligomycin showed enhanced glycolytic and glycolytic capacity of NK cells stimulated overnight with 18t15-12s, respectively. NK cells treated overnight with medium alone or a mixture of IL12, IL18, and IL-15 were used for comparison.

Example 64: Stimulation of NK cells in vivo by 2t2 and/or TGFRt15-TGFRs A series of experiments were performed to determine the effect of 2t2 constructs on immune stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solution (PBS) or 0.1, 0.4, 2, and 10 mg/kg of 2t2. Treated mice were euthanized 3 days after treatment. Spleen weights were measured and single splenocyte suspensions were prepared. Splenocyte suspensions were stained with conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies. The percentages of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells, and CD25 expression of lymphocyte subsets were analyzed by flow cytometry. FIG. 167A shows that 2t2 is effective in splenocyte proliferation based on spleen weight, especially at dose levels of 0.1-10 mg/kg. After treatment, the percentage of CD8 ⁺ T cells was higher in 2t2-treated mice compared to control-treated mice at 2 and 10 mg/kg (Fig. 167B). The percentage of NK cells was also higher in 2t2-treated mice compared to control-treated mice at all doses of 2t2 tested (FIG. 167B). In addition, 2t2 significantly upregulated CD25 expression by CD4 ⁺ T cells, but significantly upregulated CD25 expression on CD8 ⁺ T cells and NK cells after treatment at 0.4-10 mg/kg. It did not (Figure 167C).

A series of experiments were performed to determine the effect of the TGFRt15-TGFRs construct on immune stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solution (PBS) or 0.3, 1, 3, and 10 mg/kg of TGFRt15-TGFRs. Treated mice were euthanized 4 days after treatment. Spleen weights were measured and single splenocyte suspensions were prepared. Splenocyte suspensions were stained with conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies. The percentages of CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells were analyzed by flow cytometry. FIG. 168A shows that spleen weight in mice treated with TGFRt15-TGFRs increased with increasing dosage of TGFRt15-TGFRs. In addition, spleen weights in mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs were higher compared to mice treated with control solution. Figure 168B shows that the percentage of both CD8 ⁺ T cells and NK cells increased with increasing dosage of TGFRt15-TGFRs. Specifically, the percentage of CD8 ⁺ T cells was higher in mice treated with 0.3 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs compared to control-treated mice, and the percentage of NK cells was higher. was higher in mice treated with 0.3 mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs compared to control-treated mice.

A series of experiments were performed to determine the effect of TGFRt15-TGFRs or 2t2 constructs on immune stimulation in Western-fed ApoE ^−/− mice. In these experiments, 6-week-old female B6.129P2-ApoE ^tm1Unc /J mice (Jackson Laboratory) were fed 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% They were fed a western diet containing starch (TD88137, Envigo Laboratories). After 8 weeks on the Western diet, mice were injected subcutaneously with TGFRt15-TGFRs or 2t2 at 3 mg/kg. After 3 days of treatment, mice were fasted for 16 hours, after which blood samples were collected by retro-orbital venous puncture. Blood was mixed with 10 μL of 0.5 M EDTA and 20 μL of blood was taken for lymphocyte subset analysis. Erythrocytes were lysed with ACK (0.15 M _NH4Cl , 1.0 mM _KHCO3 , 0.1 mM _Na2EDTA , pH 7.4) and lymphocytes were lysed in FACS staining buffer (1% BSA in PBS). Anti-mouse CD8a and anti-mouse NK1.1 antibodies were stained for 30 minutes at 4°C. Cells were washed once and analyzed using a BD FACS Celesta. For Treg staining, ACK-treated blood lymphocytes were stained with anti-mouse CD4 and anti-mouse CD25 antibodies in FACS staining buffer for 30 minutes at 4°C. Cells were washed once, resuspended in fixation/permeabilization working solution and incubated for 60 minutes at room temperature. Cells were washed once and resuspended in permeabilization buffer. Samples were centrifuged at 300-400×g for 5 minutes at room temperature and the supernatant was discarded. The cell pellet was resuspended in the residual volume and the volume adjusted to approximately 100 μL with 1× permeabilization buffer. Anti-Fox p3 antibody was added to the cells and the cells were incubated for 30 minutes at room temperature. Permeabilization buffer (200 μL) was added to the cells and the cells were centrifuged at 300-400×g for 5 minutes at room temperature. Cells were resuspended in flow cytometry staining buffer and analyzed on a flow cytometer. Figures 169B-169C show that treatment with TGFRt15-TGFRs and 2t2 increased the percentage of NK cells and CD8 ⁺ T cells in ApoE ^−/− mice fed a Western diet. Figure 169A shows that treatment with 2t2 also increased the percentage of Treg cells.

Example 65 Induction of Immune Cell Proliferation In Vivo A series of experiments were performed to determine the effect of the 2t2 construct on immune cell stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solution (PBS) or 0.1, 0.4, 2, and 10 mg/kg of 2t2. Treated mice were euthanized 3 days after treatment. Spleen weights were measured and single splenocyte suspensions were prepared. Splenocyte suspensions were stained with conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies. The percentages of CD4 ⁺ T cells, CD8 ⁺ T cells and NK cells were analyzed by flow cytometry. FIG. 170A shows that 2t2 is effective in splenocyte proliferation based on spleen weight, especially at 0.1-10 mg/kg. The percentage of CD8 ⁺ T cells was higher compared to control treated mice at 2 and 10 mg/kg (Fig. 170B). In addition, the percentage of NK cells was higher at all doses of 2t2 tested compared to control-treated mice (FIG. 170B). These results demonstrate that 2t2 treatment was able to induce proliferation of CD8 ⁺ T cells and NK cells in C57BL/6 mice.

A series of experiments were performed to determine the effect of the TGFRt15-TGFRs construct on immune stimulation in C57BL/6 mice. In these experiments, C57BL/6 mice were treated subcutaneously with control solution (PBS) or 0.1, 0.3, 1, 3, and 10 mg/kg of TGFRt15-TGFRs. Treated mice were euthanized 4 days after treatment. Spleen weights were measured and splenocyte suspensions were prepared. Splenocyte suspensions were stained with conjugated anti-CD4, anti-CD8, and anti-NK1.1 (NK) antibodies. Cells were additionally stained for the proliferation marker Ki67. FIG. 171A shows that spleen weight in mice treated with TGFRt15-TGFRs increased with increasing dosage of TGFRt15-TGFRs. In addition, spleen weights in mice treated with 1 mg/kg, 3 mg/kg, and 10 mg/kg of TGFRt15-TGFRs were higher compared to mice treated with control solution alone. The percentages of both CD8 ⁺ T cells and NK cells increased with increasing dosage of TGFRt15-TGFRs (FIG. 171B). Finally, TGFRt15-TGFRs significantly upregulated the expression of the cell proliferation marker Ki67 on both CD8 ⁺ T cells and NK cells at all doses of TGFRt15-TGFRs tested. These results indicate that treatment with TGFRt15-TGFRs induced proliferation of both CD8 ⁺ T cells and NK cells in C57BL/6.

A series of experiments were performed to determine the effect of TGFRt15-TGFRs or 2t2 constructs on immune stimulation in Western-fed ApoE ^−/− mice. In these experiments, 6-week-old female B6.129P2-ApoE ^tm1Unc /J mice (Jackson Laboratory) were fed 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% They were fed a western diet containing starch (TD88137, Envigo Laboratories). After 8 weeks on the Western diet, mice were injected subcutaneously with TGFRt15-TGFRs or 2t2 at 3 mg/kg. After 3 days of treatment, mice were fasted for 16 hours, after which blood samples were collected by retro-orbital venous puncture. Blood was mixed with 10 μL of 0.5 M EDTA and 20 μL of blood was collected for lymphocyte subset analysis. Erythrocytes were lysed with ACK (0.15 M _NH4Cl , 1.0 mM _KHCO3 , 0.1 mM _Na2EDTA , pH 7.4) and lymphocytes were lysed in FACS staining buffer (1% BSA in PBS). Anti-mouse CD8a and anti-mouse NK1.1 antibodies were stained for 30 minutes at 4°C. Cells were washed once and resuspended in Fixation Buffer (BioLegend Catalog #420801) for 20 minutes at room temperature. Cells were centrifuged at 350 xg for 5 minutes and fixed cells were resuspended in Intracellular Staining Permeabilization Wash Buffer (BioLegend Catalog No. 421002) followed by centrifugation at 350 xg for 5 minutes. Cells were then stained with anti-Ki67 antibody for 20 minutes at room temperature. Cells were washed twice with Intracellular Staining Permeabilization Wash Buffer and centrifuged at 350×g for 5 minutes. Cells were then resuspended in FACS staining buffer. Lymphocyte subsets were analyzed using a BD FACS Celesta. As described in FIG. 172A, treatment of ApoE ^−/− mice with TGFRt15-TGFRs induced proliferation of NK cells and CD8 ⁺ T cells (Ki67 positive staining). In addition, FIG. 172B shows that treatment of ApoE ^−/− mice with 2t2 induced proliferation of NK cells and CD8 ⁺ T cells (Ki67 positive staining).

A series of experiments were performed to determine the effects of 7t15-21s plus anti-TF antibody-proliferating NK cells in NSG mice after treatment with 7t15-21s, TGFRt15-TGFRs, and 2t2. For these experiments, fresh human leukocytes were obtained from a blood bank and CD56 ⁺ NK cells were isolated with RosetteSep/human NK cell reagent (StemCell Technologies). NK cell purity was greater than 90% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). Cells were counted and supplemented with 2 mL of complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). replenishment) Resuspended at 2×10 ⁶ /mL in medium 24-well flat bottom plates. Cells were stimulated with 7t15-21s (100 nM) and anti-TF antibody (50 nM) for 15 days. Every two days, cells were resuspended at 2×10 ⁶ /mL in fresh medium containing 100 nM 7t15-21s and 50 nM anti-TF antibody. As the culture volume increased, the cells were transferred to larger volume flasks. Cells were counted using trypan blue to access fold growth. 7t15-21s+anti-TF antibody-expanded NK cells were washed three times in warm HBSS buffer (Hyclone) at 1000 RPM for 10 minutes at room temperature. 7t15-21s + anti-TF antibody-expanded NK cells were resuspended in 10×10 ⁶ /0.2 mL HBSS buffer and injected intravenously into the tail vein of NSG mice (NOD scid common gamma mice) (Jackson Laboratories). bottom. Transfected NK cells were treated with either 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 for 48 days. Upheld every hour. Engraftment and persistence of NK cells expanded with human 7t15-21s plus anti-TF antibody was measured weekly by blood staining for hCD45, mCD45, hCD56, hCD3, and hCD16 antibodies by flow cytometry (Celesta-BD Bioscience) ( Data represent 3 mice per group). FIG. 173 shows that mice with adoptively transferred 7t15-21s + anti-TF antibody expanded NK cells by adoptively transferred NK cells 7t15-21s−, TGFRt15−TGFRs−, or 2t2 compared to control treated mice. Shown are treated with induced proliferation and persistence.

Example 66: NK-mediated cytotoxicity after treatment with single- or multi-chain constructs To determine whether treatment of NK cells with TGFRt15-TGFRs enhances NK cell cytotoxicity A series of experiments were performed. In these experiments, human Daudi B lymphoma cells were labeled with CellTrace Violet (CTV) and used as tumor target cells. Mouse NK effector cells were isolated by NK1.1 positive selection using magnetic cell sorting (Miltenyi Biotec) of C57BL/6 female mouse spleens 4 days after subcutaneous treatment with 3 mg/kg TGFRt15-TGFRs. Human NK effector cells were isolated from peripheral blood mononuclear cells from human blood buffy coat using RosetteSep/human NK cell reagent (Stemcell Technologies). Target cells (human Daudi B lymphoma cells) were mixed with effector cells (either mouse NK effector cells or human NK effector cells) in the presence of 50 nM TGFRt15-TGFRs or in the absence of TGFRt15-TGFRs (control), Mouse NK cells were incubated for 44 hours and human NK cells for 20 hours at 37°C. Viability of target cells (Daudi) was assessed by analysis of 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 sample/number of viable tumor cells in sample without NK cells)×100. Figure 174 shows that mouse (Figure 174A) and human (Figure 174B) NK cells exhibit significantly greater cytotoxicity against Daudi B cells after NK cell activation with TGFRt15-TGFRs than in the absence of TGFRt15-TGFRs activation. indicate that you have

A series of experiments were performed to determine antibody-dependent cellular cytotoxicity (ADCC) of mouse and human NK cells after treatment with TGFRt15-TGFRs. In these experiments, human Daudi B lymphoma cells were labeled with CellTrace Violet (CTV) and used as tumor target cells. Mouse NK effector cells were isolated by NK1.1 positive selection using magnetic cell sorting (Miltenyi Biotec) of C57BL/6 female mouse spleens 4 days after subcutaneous treatment with 3 mg/kg TGFRt15-TGFRs. Human NK effector cells were isolated from peripheral blood mononuclear cells from human blood buffy coat using RosetteSep/human NK cell reagent (Stemcell Technologies). Target cells (Daudi B cells) were transformed into effector cells (mouse NK effector cells) in the presence of anti-CD20 antibody (10 nM rituximab, Genentech) and in the presence of 50 nM TGFRt15-TGFRs or in the absence of TGFRt15-TGFRs (control). or human NK effector cells) and incubated at 37° C. for 44 hours for mouse NK cells and 20 hours for human NK cells. Daudi B cells express the CD20 target of anti-CD20 antibodies. Target cell viability after incubation was assessed by analysis of propidium iodide-positive, CTV-labeled target cells using flow cytometry. The percentage of Daudi inhibition was calculated using the formula (1−number of viable tumor cells in experimental sample/number of viable tumor cells in sample without NK cells)×100. Figure 175 shows that mouse NK cells (Figure 175A) and human NK cells (Figure 175B) have stronger ADCC activity against Daudi B cells after activation of NK cells with TGFRt15-TGFRs than in the absence of TGFRt15-TGFRs activation. indicate that you have

A series of experiments were performed to determine the cytotoxicity of mouse NK cells activated with TGFRt15-TGFRs against senescent B16F10 melanoma cells. In these experiments, mouse NK cells were activated in vivo by injecting C57BL/6 mice with 10 mg/kg TGFRt15-TGFRs for 4 days followed by isolation of splenic NK cells. NK cells were then grown in vitro in the presence of 100 nM 2t2 for 7 days. B16F10 senescent target cells (B16F10-SNC) were labeled with CellTrace Violet (CTV) and incubated with activated mouse NK effector cells at different effector:target (E:T) ratios for 16 hours. Cells were trypsinized, washed and resuspended in complete medium containing propidium iodide (PI) solution. Cytotoxicity of TGFRt15-TGFRs/2t2-activated NK cells against senescent cell targets was accessed by flow cytometry based on PI staining of CTV-labeled cells. The findings indicate that in vivo activation of NK cells by TGFRt15-TGFRs, followed by in vivo proliferation and activation by 2t2, increased killing of senescent melanoma tumor cells by NK cells (FIG. 176). .

Example 67: A series of experiments were conducted to evaluate the anti-tumor activity of TGFRt15-TGFRs plus anti-TRP1 antibody (TA99) in combination with chemotherapy in a therapeutic melanoma mouse model of cancer, diabetes and atherosclerosis. rice field. In these experiments, C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 melanoma cells. Mice were treated with three doses of chemotherapy docetaxel (10 mg/kg) (DTX) on days 1, 4, and 7, followed by combination immunotherapy TGFRt15-TGFRs (3 mg/kg) on day 9. ) plus a single dose of anti-TRP1 antibody TA99 (200 μg). Figure 177A shows a schematic representation of the treatment regimen. Tumor growth was monitored by caliper measurements and tumor volume was calculated using the formula V=(L×W ² )/2, where L is the maximum tumor diameter and W is the perpendicular tumor diameter. Figure 177B shows that treatment with DTX + TGFRt15-TGFRs + TA99 significantly inhibited tumor growth compared to saline control and DTX-treated groups (N=10, ***p<0.001, multiple t-test analysis).

Peripheral blood analysis was performed to assess immune cell subsets in the B16F10 tumor model. In these experiments, C57BL/6 mice were injected with B16F10 cells and treated with DTX, DTX+TGFRt15-TGFRs+TA99, or saline. Blood was collected from the submandibular vein of B16F10 tumor-bearing mice 2, 5, and 8 days after immunotherapy in the DTX+TGFRt15-TGFRs+TA99 group and 11 days after tumor injection in the DTX and saline groups. RBCs were lysed in ACK lysis buffer, lymphocytes were washed and stained with anti-NK1.1, anti-CD8 and anti-CD4 antibodies. Cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figures 177C-177E show that DTX + TGFRt15-TGFRs + TA99 treatment induced an increase in the percentage of NK cells and CD8 ⁺ T cells within the tumor compared to saline- and DTX-treated groups.

On day 17, total RNA was extracted using Trizol from tumors of mice treated with saline, DTX, or DTX+TGFRt15-TGFRs+TA99. Total RNA (1 μg) was used for cDNA synthesis using the QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed on a CFX96 Detection System (Bio-Rad) using FAM-labeled pre-designed primers for senescent cell markers (F) p21 (G) DPP4 and (H) IL6. Housekeeping gene 18S ribosomal RNA was used as an internal control to normalize variations in expression levels. Expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct _target −Ct _18S . Data are expressed as fold change compared to saline controls. Figures 177F-177H show that DTX treatment induced an increase in senescent tumor cells, which was subsequently decreased after treatment with TGFRt15-TGFRs plus TA99 immunotherapy.

A series of experiments were performed to investigate the amelioration of Western diet-induced hyperglycemia in ApoE ^−/− mice by 2t2. In these experiments, 6-week-old female B6.129P2-ApoE ^tm1Unc /J mice (Jackson Laboratory) were fed 21% fat, 0.15% cholesterol, 34.1% sucrose, 19.5% casein, and 15% They were fed a western diet containing starch (TD88137, Envigo Laboratories). After 8 weeks on the Western diet, mice were injected subcutaneously with TGFRt15-TGFRs or 2t2 at 3 mg/kg. After 3 days of treatment, mice were fasted for 16 hours, after which blood samples were collected by retro-orbital venous puncture. Blood glucose levels were detected with a blood glucose meter (OneTouch UltraMini) and GenUltimate test strips using a drop of fresh blood. As shown in Figure 178A, 2t2 treatment attenuated Western diet-induced hyperglycemia (p<0.04). Plasma insulin and resistin levels were analyzed using the Mouse Rat Metabolic Array from Eve Technologies. HOMA-IR was calculated using the following formula: Homeostasis model assessment-insulin resistance = glucose (mg/dL) x insulin (mU/mL)/405. As shown in Figure 178B, 2t2 and TGFRt15-TGFRs treatment reduced insulin resistance compared to the untreated group. Both 2t2 (p<0.02) and TGFRt15-TGFRs (p<0.05) significantly reduced resistin levels compared to the untreated group, as shown in FIG. 178C, indicating that 2t2 and TGFRt15-TGFRs-induced decrease in insulin resistance (Fig. F3B).

Example 68: Induction of NK Cells to Cytokine-Induced Differentiation into Memory-Like NK Cells To assess the differentiation of NK cells into cytokine-induced memory-like NK cells (CIMK-NK cells) after stimulation with 18t15-12s conducted a series of experiments. For these experiments, fresh human leukocytes were obtained from a blood bank and CD56 ⁺ NK cells were isolated with RosetteSep/human NK cell reagent (StemCell Technologies). NK cell purity was greater than 90% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, and CD69-APCFire750 antibodies (BioLegend). Cells were counted and supplemented with 2 mL of complete medium (RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). replenishment) Resuspended at 2×10 ⁶ /mL in medium 24-well flat bottom plates. Cells were either unstimulated (“unspiked”) or 18t15-12s (100 nM), or rhIL-15 (50 ng/mL) (Miltenyi), rhIL18 (50 ng/mL) (Invivogen), and rhIL-12 (10 ng). /mL) (Peprotech) for 16 h at 37° C. and 5% CO ₂ with a single cytokine mixture (“single cytokine”). The next day, cells were harvested and washed twice with warm complete medium at 1000 RPM for 10 minutes at room temperature. Cells were resuspended at 2×10 ⁶ /mL in 2 mL complete medium containing rhIL-15 (1 ng/mL) in 24-well flat bottom plates. Every 2 days, half of the medium was replaced with fresh complete medium containing rhIL-15.

To assess changes in the memory phenotype of NK cells on day 7, cells were stained with antibodies against cell surface CD56, CD16, CD27, CD62L, NKp30, and NKp44 (BioLegend). After surface staining, cells were washed (1500 RPM at room temperature) in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). 5 minutes). After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience). Figure 179 shows that incubation of NK cells with 18t15-12s increased the percentage of CD16 ⁺ CD56 ⁺ NK cells expressing CD27, CD62L, and NKp44 and increased the levels of NKp30 in CD16 ⁺ CD56 ⁺ NK cells (MFI ) increased.

Example 69. Upregulation of CD44 memory T cells C57BL/6 mice were treated subcutaneously with TGFRt15-TGFRs or 2t2. Treated mice were euthanized and single splenocyte suspensions were prepared 4 days (TGFRt15-TGFRs) or 3 days (2t2) after treatment. Prepared splenocytes were stained with fluorochrome-conjugated anti-CD4, anti-CD8, and anti-CD44 antibodies and the percentage of CD44- ^rich T cells among CD4 ⁺ T cells or CD8 ⁺ T cells was analyzed by flow cytometry. The results show that TGFRt15-TGFRs and 2t2 upregulated the expression of memory marker CD44 on CD4 ⁺ and CD8 ⁺ T cells (FIG. 180). These findings indicate that TGFRt15-TGFRs and 2t2 were able to induce differentiation of mouse T cells into memory T cells.

Example 70: Improving Texture and/or Appearance and/or Hair To investigate the effect of 2t2 on hair regrowth, the hair on the dorsal side of C57BL6/J mice (Jackson Laboratory) was first clipped short, followed by hair clippers. A depilatory cream (Nair) was applied to the shaved area for 30 seconds and then wiped clean. Four hours later, 2t2 (3 mg/kg, single dose), low-dose recombinant IL-2 (25000 IU, 5 consecutive days, 1 dose/day), or PBS were administered subcutaneously. Mice were observed for skin pigmentation associated with hair regrowth, imaged and analyzed using ImageJ software. FIG. 181A shows skin pigmentation 10 days after depilation in mice treated with PBS, 2t2, or IL-2. Figure 181B shows the pigmentation rate of each group of mice after 10 days of treatment, analyzed using Image J software. The results showed that treatment of mice with 2t2 or IL-2 enhanced hair regrowth after depilation compared to mice treated with PBS.

After the dorsal hair of C57BL6/J mice (Jackson Laboratory) was first clipped short, depilatory cream (Nair) was applied to the shaved area for exactly 30 seconds and then wiped clean. Four hours later, 2t2 (3 mg/kg, single dose), low-dose recombinant IL-2 (25000 IU, 5 consecutive days, 1 dose/day), or PBS were administered subcutaneously. Mice were observed for skin pigmentation associated with hair regrowth, imaged and analyzed using ImageJ software. Figure 182 shows skin pigmentation 14 days after depilation in mice treated with PBS, 2t2, or IL-2. The results showed that treatment of mice with 2t2 or IL-2 enhanced hair regrowth after depilation compared to mice treated with PBS.

Example 71 Tissue Factor Coagulation Assay After Treatment with Single- or Multi-Chain Chimeric Polypeptides A series of experiments were performed to assess blood clotting after treatment with single- or multi-chain chimeric polypeptides. To initiate the blood clotting cascade pathway, tissue factor (TF) binds to factor VIIa (FVIIa) to form the TF/FVIIa complex. The TF/FVIIa complex then binds to Factor X (FX), converting FX to FXa.

Factor VIIa (FVIIa) Activity Assay One assay for measuring blood clotting involves measuring Factor VIIa (FVIIa) activity. This type of assay requires the presence of tissue factor and calcium. TF/FVIIa complex activity can be measured using small substrates or natural protein substrates such as Factor X (FX). Phospholipids are also required for TF/FVIIa activity when FX is used as a substrate. In this assay, FVIIa activity is determined using the 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 (such as the TF/FVIIa complex).

In these experiments, the following groups were tested: the 219-amino acid extracellular domain of the tissue factor domain ( _TF219 ), multi-chain chimeric polypeptides with wild-type tissue factor domains, and multi-chain chimeric polypeptides with mutant tissue factor domains. FVIIa activity was compared. A chimeric polypeptide containing a mutant tissue factor molecule was constructed by mutation to the TF domain at amino acid sites: Lys20, Ile22, Asp58, Arg135, and Phe140.

To assess the activity of FVIIa, FVIIa and TF ₂₁₉ or TF _219- containing multichain chimeric polypeptides were mixed in a total volume of 70 μL in all wells of a 96-well ELISA plate at equimolar concentrations (10 nM). After a 10 minute incubation at 37° C., 10 μL of 8 mM S-2288 substrate was added to initiate the reaction. After that, incubation was continued for 20 minutes at 37°C. Finally, color change was monitored by reading the absorbance at 405 nm. OD values for various TF/VIIa complexes are shown in Tables 1 and 2. Table 1 shows a comparison of TF ₂₁₉ , 21t15-21s wild type (WT), and 21t15-21s mutant (Mut). Table 2 shows a comparison of TF ₂₁₉ , 21t15-TGFRs wild-type (WT), and 21t15-TGFRs mutant (Mut). These data demonstrate that TF _219- containing multichain chimeric polypeptides (eg, 21t15-21s-WT, 21t15-21s-Mut, 21t15-TGFRS-WT, and 21t15-TGFRS) when chromogenic S-2288 is used as a substrate. -Mut) has lower FVIIa activity than _TF219 . Notably, multi-chain chimeric polypeptides containing the _TF219 mutation exhibited much lower FVIIa activity when compared to multi-chain chimeric polypeptides containing wild-type _TF219 .

(Table 1) FVIIa activity

(Table 2) FVIIa activity

Factor X (FX) Activation Assay An additional assay for measuring blood clotting involves measuring activation of Factor X (FX). Briefly, TF/VIIa activates blood coagulation factor X (FX) to factor Xa (FXa) in the presence of calcium and phospholipids. TF ₂₄₃ , which contains the transmembrane domain of TF, has much higher activity than TF ₂₁₉ , which does not contain the transmembrane domain, in activating FX to FXa. TF/VIIa-dependent activation of FX is determined by measuring FXa activity using the FXa-specific chromogenic substrate S-2765 (Diapharma, West Chester, 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 by multichain chimeric polypeptides (18t15-12s, mouse (m) 21t15, 21t15-TGFRs, and 21t15-7s) was compared to a positive control (Innovin) or _TF219 . TF ₂₁₉ (or TF _219- containing multichain chimeric polypeptide)/FVIIa complexes were mixed in 50 μL volumes of equimolar concentrations (0.1 nM each) in round-bottom wells of 96-well ELISA plates, followed by 10 μL of 180 nM. FX was added. FX was converted to FXa during a 15 min incubation at 37° C., after which 8 μL of 0.5 M EDTA (which chelates calcium and thus terminates FX activation by TF/VIIa) was added to each well. to stop FX activation. 10 μL of 3.2 mM S-2765 substrate was then added to the reaction mixture. The plate absorbance was immediately measured at 405 nm and recorded as the absorbance at time 0. Plates were then incubated for 10-20 minutes at 37°C. Color change was monitored by reading absorbance at 405 nm after incubation. Results of FX activation as measured by FXa activity using the chromogenic substrate S-2765 are shown in FIG. Innovin, a commercial prothrombin reagent containing lipidated recombinant human TF ₂₄₃ , was used as a positive control for FX activation in this experiment. Innovin was reconstituted with purified water to approximately 10 nM _TF243 . A 0.1 nM TF/VIIa complex was then made by mixing an equal volume of 0.2 nM FVIIa with 0.2 nM Innovin. Innovin showed very potent FX activating activity, whereas TF ₂₁₉ and TF _219- containing multichain chimeric polypeptides had very low FX activating activity, indicating that TF ₂₁₉ activated the natural substrate FX in vivo. It was confirmed that it was not active in the TF/FVIIa complex to do so.

Prothrombin Time Test A third assay for measuring blood clotting is the prothrombin time (PT) test, which measures blood clotting activity. Here, PT tests were performed using commercially available normal human plasma (Ci-Trol Coagulation Control, Level I). For the standard PT test, the clotting reaction was initiated by addition of Innovin, a lipidated recombinant human TF ₂₄₃ , in the presence of calcium. Clotting times were monitored and reported by a START PT Analyzer (Diagnostica Stago, Parsippany, N.J.). The PT assay was performed by prewarming 0.2 mL of various Innovin dilutions in PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6 mM CaCl ₂ , 0.1% BSA) at 37°C. .By injecting into a cuvette containing 1 mL of normal human plasma. In the PT assay, a shorter PT time (clotting time) indicates higher TF-dependent coagulation activity, and a longer PT (clotting time) means lower TF-dependent coagulation activity.

As seen in Figure 184, the addition of different amounts of Innovin (e.g., Innovin reconstituted in purified water equivalent to 10 nM lipidated recombinant human TF ₂₄₃ is considered 100% Innovin) to the PT assay. A dose-response relationship was demonstrated, with lower TF ₂₄₃ concentrations leading to longer PT times (lower clotting activity). For example, 0.001% Innovin had a PT time of over 110 seconds, which was about the same as buffer alone.

In another experiment, PT studies were performed on multichain chimeric polypeptides containing TF ₂₁₉ and 18t15-12s, 7t15-21s, 21t15-TGFRs-WT, and 21t15-TGFRs-Mut. Figure 185 shows that TF ₂₁₉ and TF _219- containing multichain chimeric polypeptides (100 nM concentration) have long PT times and very low or no clotting activity.

To assess whether incubating a multichain chimeric polypeptide in the presence of other cells (32Dβ or human PBMC) that have receptors for the cytokine components of the multichain chimeric polypeptide affects clotting time in the PT assay We also conducted a study on Cells expressing IL-15 receptor (32Dβ cells) or IL-15 and IL-21 receptors (PBMC) mimic IL-15-containing multichain chimeric polypeptides as cellular FVIIa receptors To test for binding to native TF, TF _219- containing multichain chimeric polypeptides (at a concentration of 100 nM each molecule) were diluted in PT assay buffer and plated with 32Dβ cells (2 x 10 ⁵ cells/mL) or PBMCs. (1×10 ⁵ cells/mL) for 20-30 minutes at room temperature. PT assays were then performed as described above. Figures 186 and 187 demonstrate that TF ₂₁₉ and TF ₂₁₉ -containing multi-chain chimeric polypeptides mixed with 32Dβ cells (Figure 186) or PBMCs (Figure 187) at a final concentration of 100 nM showed that 0.001-0.01% Innovin (equivalent to 0.1 pM to 1.0 pM TF ₂₄₃ ). Expressed as a percentage of relative TF ₂₄₃ activity, TF _219- containing multichain chimeric polypeptides had 100,000- to 1,000,000-fold lower TF-dependent coagulant activity compared to Innovin. This suggests that TF _219- containing multichain chimeric polypeptides had very low TF-dependent coagulant activity, even while the molecule was bound to intact cell membrane surfaces such as 32Dβ or PBMC. showed that it did not have

Example 72: Characterization of 7t15-21s137L (long version) The nucleic acid sequence (including signal peptide sequence) of the 7t15 construct is as follows (SEQ ID NO:210).
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL7)
GATTGCGACATCGAGGGCAAGGACGGCAAGCAGTACGAGAGCGTGCTGATGGTGTCCATCGACCAGCTGCTGGACAGCATGAAGGAGATCGGCTCCAACTGCCTCAACAACGAGTTCAACTTCTTCAAGCGGCCACATCTGCGACGCCAACAAGGAGGGCATGTTCCT GTTCAGGGCCGCCAGGAAACTGCGGCAGTTCCTGAAGATGAACTCCACCGGCGACTTCGACCTGCACCTGCTGAAGGTGTCCGAGGGCACCACCATCCTGCTGAACTGCACCGGACAGGTGAAGGGCCGGAAACCTGCTGCTCTGGGAGAGGCCCAACCCACCAAGAGCCTGGAGGAGA ACAAGTCCCTGAAGGAGCAGAAGAAGCTGAACGACCTGTGCTTCCTGAAGAGGCTGCTGCAGGAGATCAAGACCTGCTGGAACAAGATCCTGATGGGCACCAAGGAGCAT
(human tissue factor 219)
AGCGGCCAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAACCATCCTCGAATGGGAACCCAAACCCGTTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCG ATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCTTTATACGAGAACAGCCCCGAATTTACCCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCA CAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTAATCGACG TGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAGGGCGAGTTCCGGGAG
(human IL-15)
AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTACTGGAGCTGCCAAGTTATCTCTTTAGAGAGCGGGAGACGCTAGCAT CCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCCAAGGAGTGCGAAGAGCTGGAGGGAGAAGAACATCAAGGAGTTCTGCAATCCTTTGTGCCACATTGTCCAGATGTTCATCAATACCTCC

The amino acid sequence (including leader sequence) of the 7t15 fusion protein is as follows (SEQ ID NO:209).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL7)
DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDHLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWN KILMG TKEH
(human tissue factor 219)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(human IL-15)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

The nucleic acid sequence (including signal peptide sequence) of the 21s137L construct is as follows (SEQ ID NO:331).
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(human IL-21)
CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCG CCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCCTCCACAAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCCTGCTGCAGAAGAT GATCCATCAGCACCTGTCCTCCAGGACCCAGGCTCCGAGGACTCC
(Human IL-15Rα sushi domain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG
((G4S)3 linker)
GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGGCGGAGGATCT
(human CD137L)
CGCGAGGGTCCCGAGCTTTCGCCCCGACGATCCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCGCAGCTGGTGGCCCAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCCAGGCCTGGCAGGGCGTGTCCCTGACGGGGGGC CTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTG GCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAGTCTTGG GACTCTTCCGGGTGACCCCCGAAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA

The amino acid sequence (including leader sequence) of the 21s137L fusion protein is as follows (SEQ ID NO:332).
(signal peptide)
MKWVT FISLLFLFS SAYS
(human IL-21)
QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS
(Human IL-15Rα sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
((G4S)3 linker)
GGGGSGGGGSGGGGGS
(human CD137L)
REGPELSPDDPAGLLDLLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSSVSLALHLQPLRSAAGAAALALTVDLLPPASSEARNSAFGFQGRLLHLSAGQRLGV HLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSSE

The following experiments were performed to assess whether the CD137L portion in 7t15-21s137L is intact to bind CD137 (4.1BB). On day 1, 96-well plates were coated overnight with 100 μL (2.5 μg/mL) of GAH IgG Fc (G-102-C, R&D Systems) in R5 (coating buffer). On day two, plates were washed three times and blocked with 300 μL of 1% BSA in PBS for 2 hours at 37°C. 10 ng/mL 4.1BB/Fc (838-4B, R&D Systems) was added at 100 μl/well for 2 hours at room temperature. After 3 washes, 7t15-21s137L (long version) or 7t15-21s137L (short version) from 10 nM or recombinant human 4.1BBL from 180 ng/mL was added at a 1/3 dilution and then added at 4°C. was incubated overnight at. On day 3, plates were washed three times and 500 ng/mL biotinylated goat anti-human 4.1BBL (BAF2295, R&D Systems) was applied at 100 μL/well followed by incubation at room temperature for 2 hours. Plates were washed three times and incubated with 0.25 μg/mL HRP-SA (Jackson Immune Research) at 100 μL/well for 30 minutes. Plates were then washed three times and incubated with 100 μL ABTS for 2 minutes at room temperature. Results were read at 405 nm. As shown in Figure 188, both 7t15-21s137L (long version) and 7t15-21s137L (short version) are 4.1BB /Fc (dark diamonds and gray squares). 7t15-21s137L (long version) (dark diamonds) interacted better with 4.1BB/Fc compared to 7t15-21s137L (short version) (grey squares).

The following experiments were performed to assess whether the components IL7, IL21, IL15, and 4.1BBL in 7t15-21s137L (long version) are intact for detection by individual antibodies using ELISA. . A 96-well plate was coated with 100 μL (4 μg/mL) of anti-TF (human IgG1) in R5 (coating buffer) and incubated for 2 hours at room temperature. Plates were washed three times and blocked with 100 μL of 1% BSA in PBS. Purified 7t15-21s137L (long version) was added at a 1/3 dilution from 10 nM, followed by incubation for 60 minutes at room temperature. Plates were washed 3 times and added with 500 ng/mL biotinylated anti-IL7 (506602, R&D Systems), 500 ng/mL biotinylated anti-IL21 (13-7218-81, R&D Systems), 50 ng/mL biotinylated anti-IL15 (506602, R&D Systems). BAM247, R&D Systems) or 500 ng/mL biotinylated goat anti-human 4.1BBL (BAF2295, R&D Systems) was added to each well and incubated for 60 minutes at room temperature. Plates were washed three times and incubated with 0.25 μg/mL HRP-SA (Jackson ImmunoResearch) at 100 μL/well for 30 minutes at room temperature. Plates were washed 4 times and incubated with 100 μL ABTS for 2 minutes at room temperature. Absorbance results were read at 405 nm. As shown in Figures 189A-189D, components including IL7, IL21, IL15, 4.1BBL in 7t15-21s137L (long version) were detected by individual antibodies.

The following experiments were performed to assess the activity of IL15 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 IL2Rαβγ-expressing CTLL2 cells was compared to that of recombinant IL15. IL15-dependent CTLL2 cells were washed five times with IMDM-10% FBS and seeded in wells at 2×10 ⁴ cells/well. Serially diluted 7t15-21s137L (long version), 7t15-21s137L (short version), or IL15 were added to the cells. Cells were incubated for 3 days at 37°C in a _CO2 incubator. Cell proliferation was detected by adding 20 μL of PrestoBlue (A13261, ThermoFisher) to each well on day 3 and incubating for an additional 4 hours at 37° C. in a CO ₂ incubator. Raw absorbance at 570-610 nm was read on a microtiter plate reader. As shown in Figure 190, 7t15-21s137L (long version), 7t15-21s137L (short version), and IL15 all promoted CTLL2 cell proliferation. The _EC50s of 7t15-21s137L (long version), 7t15-21s137L (short version) and IL15 are 51.19 pM, 55.75 pM and 4.947 pM, respectively.

Example 73: Induction of Treg cells by 2t2 Peripheral blood mononuclear cells (PBMC) of a healthy donor (donor 163) were isolated from 5 mL of whole blood buffy coat by Ficoll Paque Plus (GE17144003). PBMC were then lysed with ACK to remove red blood cells. Cells were washed with IMDM-10% FBS and counted. 1.8×10 ⁶ cells (100 μL/tube) were seeded in flow tubes and 50 μL of descending 2t2 or IL2 (15000, 1500, 150, 15, 1.5, 0.15, or 0 pM) and 50 μL of Incubated with prestaining antibodies (anti-CD8-BV605 and anti-CD127-AF647). Cells were incubated in a water bath at 37°C for 30 minutes. Stimulation was stopped by adding 200 μL of pre-warmed BD Phosflow Fix Buffer I (Cat#557870, Becton Dickinson Biosciences) in a water bath at 37° C. for 10 minutes. Cells (4.5×10 ⁵ cells/100 μL) were transferred to a V-shaped 96-well plate, spun down, and then spun down with 100 μL of BD Phosflow Perm Buffer III (catalog number BD Biosciences) precooled to −20° C. Permeabilize for 30 minutes on ice. Cells were then extensively washed twice with 200 μL of FACS buffer and tested with a panel of fluorescent antibodies (anti-CD25-PE, CD4-PerCP-Cy5.5, CD56-BV421, CD45RA-PE-Cy7, and pSTAT5a-AF488). ) to distinguish different lymphocyte subpopulations and assess pSTAT5a status. Cells were spun down and resuspended in 200 μL FACS buffer for FACSCelesta analysis. As shown in Figure 191A, 6 pM of 2t2 was sufficient to induce phosphorylation of Stat5a in CD4 ⁺ CD25 ^high T _reg cells, whereas 43.11 pM IL-2 reduced Stat5a in the same population of lymphocytes. was required to induce the phosphorylation of In contrast, 2t2 was less active (FIG. 191B) or equally active in inducing Stat5a phosphorylation in CD4 ⁺ CD25 ⁻ T _con and CD8 ⁺ T _con cells when compared to IL2. (Fig. 191C). These results demonstrate that 2t2 is superior to IL2 in activating T _regs in human PBMCs, and that 2t2 is superior to IL2 in activating T _regs in human blood lymphocyte pStat5a responses. and to demonstrate that it enhances selectivity.

Example 74. Improving Hair Growth Using Single-Chain Chimeric Polypeptides Seven-week-old C57BL6/J mice were shaved dorsally and depilated using a commercial depilatory cream. Mice were injected subcutaneously with a single dose of 2t2 or a low dose of commercially available recombinant IL-2 on the same day, followed by daily dosing for an additional 4 days. Untreated mice were used as controls. On day 10, mice were sacrificed and skin sections of shaved areas were prepared. Representative H&E staining of skin sections of C57BL6J mice 10 days after depilation are shown in Figures 192A-192E. Figure 192A shows only control mice that were shaved followed by depilation, Figure 192B shows mice that received a low dose of IL-2 (1 mg/kg) after depilation, and Figures 192C-192E show: Shown are mice dosed with 0.3 mg/kg (Figure 192C), 1 mg/kg (Figure 192D), and 3 mg/kg (Figure 192E) of 2t2 after depilation. Black arrows indicate anagen hair follicles that later extend into the dermis and promote hair growth. Figure 194 shows the total number of anagen follicles counted per 10 fields for each treatment group. In summary, the data show that the 2t2 molecule resulted in an increase in the number of anagen hair follicles compared to depilation alone. This effect was also dose dependent.

Example 75 Differentiation of Immune Cells into Memory-Like Immune Cells Fresh human leukocytes were obtained from a blood bank and CD56 ⁺ NK cells were isolated with RosetteSep/human NK cell reagent (StemCell Technologies). The purity of NK cells was >70% and confirmed by staining with CD56-BV421, CD16-BV510, CD25-PE, CD69-APCFire750 (BioLegend). Cells were counted in RPMI 1640 supplemented with 2 mM L-glutamine (Thermo Life Technologies), antibiotics (penicillin, 10,000 units/mL, 10,000 μg/mL; Thermo Life Technologies), and 10% FBS (Hyclone). Resuspended at a density of 2×10 ⁶ cells/mL in medium (Gibco). Cells (1 mL) were transferred to 24-well flat bottom plates and grown in culture for 14 days with no treatment or 7t15-21s plus anti-tissue factor (TF) antibody (IgG1) (50 nM). Cells were supplemented with fresh 7t15-21s plus anti-TF antibody (IgG1) (50 nM) to maintain a cell density of approximately 1×10 ⁶ cells/mL.

Non-proliferating NK cells for treatment groups were used as a positive control for complete DNA methylation levels (data not shown). NK cells were pelleted (1×10 ⁶ ) and genomic DNA (nDNA) was isolated using the QIAamp UCP DNA Micro Kit (Qiagen). 500 ng of purified nDNA was subjected to sodium bisulfite treatment using the EZ DNA Methylation-Direct kit (Zymo Research) according to the manufacturer's protocol. Bisulfite treatment produces methylation-dependent changes in DNA, converting demethylated cytosines to uracil, but leaving methylated cytosines unchanged. Bisulfite-treated nDNA (10-50 ng) was treated with forward primer IFNG127F (5′-ATGGTATAGGTGGGTATAATGG-3′) and biotinylated reverse primer IFNG355R-bio (biotin-5′-CAATATATACACCTCTCCTCTAACTAC-3′) (GENEWIZ). Two CpG sites (CpG-186 and CpG-54, for the transcription start site TSS), known to be highly regulated by DNA methylation in T cells using the Pyrmark PCR kit (Qiagen) A 228 bp region of the IFNγ promoter containing the position ) was PCR amplified as a template. The PCR conditions were 95°C for 15 minutes, 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 60 seconds, followed by 48 cycles of 72°C for 10 minutes. The integrity and quality of PCR amplification products were visualized on 1.2% TAE agarose gels. The DNA methylation status of these two CpG sites is determined by pyrosequencing, the gold standard technique for quantitatively measuring DNA methylation at single CpG sites. Pyrosequencing reactions were performed using DNA sequencing primers C186-IFNG135F (5′-GGTGGGTATAATGGG-3′) (SEQ ID NO: 333) and C54-IFNG261F (5′-ATTATTTTATTTTAAAAAATTTGTG-3′) specific for CpG sites 186 and 54, respectively. (SEQ ID NO: 334) was performed at the Genetic Resources Core Facility at Johns Hopkins University. Commercially available unmethylated and methylated DNA (Zymo Research) were used as controls for DNA methylation. The methylation percentages of two CpG sites (-186 and -54) were pooled for each treatment. The percent difference in DNA methylation was calculated relative to the level of DNA methylation at the two CpG sites observed in unexposed NK cells.

Analysis of the DNA methylation status of these two IFNγ CpG sites revealed higher levels of DNA demethylation in NK cells supported by 7t15-21s + anti-TF antibody compared to unexposed NK cells. (Fig. 194). These 7t15-21s+ anti-TF antibody-bearing NK cells exhibited a 47.70%±11.76 difference in DNA methylation (ie, demethylation) compared to unexposed NK cells. DNA methylation levels at these two IFNγ CpG sites correlated with increased expression of IFNγ following treatment with 7t15-21s plus anti-TF antibody. These data demonstrate that long-term exposure of NK cells (expansion for 14 days in culture) with a combined regimen of 7t15-21s plus an anti-TF antibody results in DNA demethylation of two hypomethylated IFNγ CpG sites (−186 and −54). and that 7t15-21s plus anti-TF antibody (IgG1) epigenetically reprograms gene expression of IFNγ through DNA demethylation of CpG sites, transforming NK cells into innate immune memory NK cells. suggest that the interconversion of

Example 76: Chemotherapy induces p21 CIP1 p21 senescence-related gene expression in C57BL/6 mice Chemotherapy induces p21 ^CIP1 p21 senescence-related gene expression in C57BL/6 mice. FIG. 203A is a schematic diagram showing a treatment regimen. C57BL/6 mice were treated with chemotherapy docetaxel (DTX) (10 mg/kg) three times on days 1, 4 and 7. Mice were sacrificed on day 9 and lung and liver tissues were collected to assess markers of senescence. Figures 203B and 203C show expression of p21 ^CIP1 p21 in lung (B) and liver (C) tissues, respectively. Lung and liver tissues were homogenized using a mortar and pestle in liquid nitrogen. Homogenized tissue was transferred to a new Eppendorf tube containing 1 mL of Trizol (Thermo Fischer). Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers p21 ^CIP1 p21 purchased 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 variations in expression levels. Expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct _target −Ct _18S . As shown in Figures 203A-203C, the senescence marker p21 ^CIP1 p21 was induced in lung and liver tissues of mice treated with docetaxel.

Example 77: Immunophenotype and Cell Proliferation After Treatment with IL-15-Based Agents (Day 3 Post-Treatment)
Mouse blood was prepared to evaluate different subsets of immune cells after treatment with TGFRt15-TGFRs. Six-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into groups as follows: saline control group (n=6), docetaxel group (n=6), docetaxel in TGFRt15-TGFRs group (n=6), docetaxel in IL-15SA group (n=6) = 6). An IL-15 superagonist (IL-15SA) was constructed and administered as previously described (Zhu et al., J. Immunol. 183(6):3598-3607, 2009). Three doses of docetaxel (10 mg/kg) on days 1, 4, and 7 induced senescence in mice. On day 8, mice were treated subcutaneously with either PBS or TGFRt15-TGFRs (3 mg/kg) or IL-15SA (0.2 mg/kg). Mouse blood was collected from the submandibular vein 3 days after treatment with EDTA-containing tubes. Whole blood was centrifuged at 3000 RPM for 10 minutes in a microcentrifuge to collect plasma. Plasma was stored at −80° C. and whole blood was processed for immune cell phenotyping by flow cytometry. Whole blood was dissolved in ACK buffer for 5 minutes at room temperature. Cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells in the blood, cell surface CD4, CD45, CD8, and NK1.1 (Biolegend) were stained for 30 minutes at room temperature. After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). Cells were treated with permeabilization buffer (Invitrogen) for 20 min at 4° C. and then washed with Perm buffer (Invitrogen). Cells were then stained for intracellular markers (Ki67) and FoxP3 for 30 minutes at room temperature. After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience). These data indicate that the IL-15-based agonists TGFRt15-TGFRs and IL-15SA can stimulate and promote the expansion and proliferation of NK and CD8 ⁺ T cells after docetaxel treatment (Figure 204).

Example 78: TGFRt15-TGFRs treatment reduces senescence-related gene expression in C57BL/6 mice Chemotherapy-induced senescence- related gene expression was significantly reduced in lung and liver of C57BL/6 mice with TGFRt15-TGFRs. C57BL/6 mice were treated with chemotherapy docetaxel (10 mg/kg) three times on days 1, 4 and 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 were used as controls. TGFRt15-TGFRs were administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.2 mg/kg. Three days after study drug treatment, mice were sacrificed and lungs and livers were collected. Graphs 205A-205C show p21 ^CIP1 p21 and CD26 expression in lung (A and B) tissues and p21 ^CIP1 p21 expression in liver (C) tissues, respectively. Lung and liver tissues were homogenized using a mortar and pestle in liquid nitrogen. Homogenized tissue was transferred to a new Eppendorf tube containing 1 mL of Trizol (Thermo Fischer). Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed on the CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers p21 ^CIP 1p21 and CD26 purchased 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 variations in expression levels. Expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct _target −Ct _18S .

As shown in Figures 205A-205C, therapy-induced senescence markers p21 ^CIP1 p21 were significantly reduced in lung and liver tissues of mice treated with TGFRt15-TGFRs. The therapy-induced senescence marker CD26 was also significantly reduced in lung tissue from mice treated with TGFRt15-TGFRs.

Example 79: Blood of immunophenotypic mice after treatment with IL-15-based agents showed IL-15-based agents: TGFRt15-TGFRs, IL-15 superagonist (IL-15SA), and IL-15- were prepared to evaluate various subsets of immune cells after treatment with IL-15 fusions of D8N mutants that knocked out the 15 activity (TGFRt15*-TGFRs). Six-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into groups (n=6/group) and treated with: 1) PBS (saline) control, 2) docetaxel, 3) docetaxel with TGFRt15-TGFRs, 4) docetaxel with IL-15SA. 5) docetaxel with IL-15 variants (TGFRt15*-TGFRs) and 6) docetaxel with IL-15 superagonist (IL-15SA) and TGFRt15*-TGFRs. Three doses of docetaxel (10 mg/kg) on days 1, 4, and 7 induced senescence in mice. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs, IL-15SA, or a combination of the above. TGFRt15-TGFRs and TGFRt15*-TGFRs were administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.05 mg/kg. Mouse blood was collected into EDTA tubes from the submandibular vein on day 3 after study drug treatment. Whole blood was centrifuged at 3000 RPM for 10 minutes in a microcentrifuge to collect plasma. Plasma was stored at −80° C. and whole blood was processed for immune cell phenotyping by flow cytometry. Whole blood was lysed with ACK buffer at 37° C. for 5 minutes. Cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells in blood, cell surface CD4, CD45, CD19CD8, and NK1.1 (Biolegend) were stained for 30 minutes at room temperature (RT). After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). Cells were treated with permeabilization buffer (Invitrogen) for 20 min at 4° C. and then washed with Perm buffer (Invitrogen). Cells were then stained for an intracellular marker (Ki67) for 30 minutes at room temperature. After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience) (Figures 206 and 207).

These data demonstrate that the IL-15-based agents TGFRt15-TGFRs and IL-15SA can stimulate and promote NK and CD8 ⁺ T cell expansion and proliferation after docetaxel treatment. Fusion proteins lacking IL-15 activity (ie, TGFRt15*-TGFRs) did not result in increased expansion and proliferation of NK and CD8 ⁺ T cells.

Example 80 Evaluation of Senescence Markers p21CIP1p21 and CD26 in Lung and Liver Tissues Markers of cellular senescence were evaluated in tissues of normal mice after administration of chemotherapy and study treatments. Six-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into 6 groups and treated with: 1) PBS (saline) control (n=5), 2) docetaxel (n=8), 3) docetaxel with TGFRt15-TGFRs (n=8). 4) docetaxel with IL15SA (n=8), 5) docetaxel with IL-15 variants (TGFRt15*-TGFRs) (n=8) and 6) IL-15 superagonist (IL-15SA) with TGFRt15*. - Docetaxel with TGFRs (n=6). Three doses of docetaxel (10 mg/kg) on days 1, 4, and 7 induced senescence in mice. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs, IL-15SA, or the following combinations. TGFRt15-TGFRs and TGFRt15*-TGFRs were administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.05 mg/kg. Mouse tissues were prepared to assess different markers of senescence. Mice were euthanized 7 days after study drug treatment and liver and lung tissues were collected and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. The samples were homogenized using a mortar and pestle in liquid nitrogen. Homogenized tissue was transferred to a new Eppendorf tube containing 1 mL of Trizol (Thermo Fischer). Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions and 1 μg of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in expression levels. Expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct _target −Ct _18S . As shown in FIGS. 208A-208C, senescence markers p21 and CD26 were induced in lung [(A) and (B), respectively] tissues of mice treated with docetaxel, and p21 ^CIP1 p21 was induced in liver (C) tissue. rice field. Pulmonary senescence markers p21 ^CIP1 p21 and CD26 and liver senescence markers ^{p21 CIP1} p21 were reduced in mice treated with TGFRt15-TGFRs, IL-15SA, and the combination of IL-15SA and TGFRt15*-TGFRs mutants. However, the lungs of mice treated with TGFRt15*-TGFRs mutants failed to eliminate these tissue markers of senescence. These results indicate that IL-15 activity is important for elimination of TIS senescent cells.

Example 81: Immunophenotypes after treatment with TGFRt15-TGFRs To assess different subsets of immune cells after treatment with TGFRt15-TGFRs, mouse blood was prepared. 76-week-old C57BL/6 aged mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups as follows: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). On day 0, mice were treated subcutaneously with PBS or TGFRt15-TGFRs at a dosage of 3 mg/kg. Mouse blood was collected from the submandibular vein in EDTA-containing tubes four days after the first dose of study treatment. Whole blood was centrifuged at 3000 RPM for 10 minutes in a microcentrifuge to collect plasma. Plasma was stored at −80° C. and blood was processed for immune cell phenotyping by flow cytometry. Whole blood was lysed with ACK buffer for 5 minutes at room temperature. Cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells in blood, cell surface CD4, CD45, CD19CD8 and NK1.1 (Biolegend) were stained for 30 min at room temperature (RT). After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). Cells were treated with permeabilization buffer (Invitrogen) for 20 min at 4° C. and then washed with Perm buffer (Invitrogen). Cells were then stained for an intracellular marker (Ki67) for 30 minutes at room temperature. After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figure 195, the percentage of CD8 ⁺ T cells and proliferation of CD8 ⁺ T cells measured by Ki67 were significantly increased 4 days after the first dose of TGFRt15-TGFRs. In addition, as shown in Figure 196, an increase in NK cells and proliferation of NK cells were also observed. A significant decrease in CD19 ⁺ cells was observed after the first dose of TGFRt15-TGFRs. These results demonstrate that subcutaneous administration of a single dose of TGFRt15-TGFRs can stimulate immune cells such as CD8 ⁺ T cells and NK cells to proliferate in the blood of aged mice.

Example 82: TGFRt15-TGFRs Reduce Senescence-Associated β-Gal from Liver and Lung Tissue Liver and lungs of mice were prepared to assess senescence-associated β-gal in tissues after treatment with TGFRt15-TGFRs. bottom. 76-week-old C57BL/6 aged mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups as follows: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). On days 0 and 10, mice were treated subcutaneously with PBS or TGFRt15-TGFRs at a dosage of 3 mg/kg. Seven days after the second dose of study treatment, mice were euthanized and livers and lungs were collected, homogenized with PBS containing 2% PBS and filtered through a 70 micron filter to obtain single cell suspensions. I got the liquid. Cells were spun down and resuspended in 5 mL RPMI containing 0.5 mg/mL collagenase IV and 0.02 mg/mL DNAse in a 14 mL round bottom tube. Cells were then shaken on an orbital shaker at 37° C. for 1 hour. Cells were washed twice with RPMI. Cells were cultured in 2 mL complete medium (supplemented with RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). inside Resuspended at 2×10 ⁶ /mL in 24-well flat bottom plates and incubated at 37° C., 5% CO ₂ for 48 hours. Cells were harvested and washed once with warm complete medium at 1000 rpm for 10 minutes at room temperature. Cell pellets were resuspended in 500 μL of fresh medium containing 1.5 μL of senescence dye per tube. Cells were then further incubated at 37° C., 5% CO ₂ for 1-2 hours and washed twice with 500 μL of wash buffer. Cell pellets were analyzed by flow cytometry (Celesta-BD Bioscience) immediately after resuspending cells in 500 μL of wash buffer.

As shown in Figure 197, the percentage of senescence-associated β-gal ⁺ cells decreased 7 days after the second dose of TGFRt15-TGFRs. These results demonstrate that TGFRt15-TGFRs can reduce senescence-associated β-gal in tissues of aged mice.

Example 83: Senescence Markers CD26, IL-1α, p16INK4 and p21CIP1 in Kidney, Skin, Liver and Lung Tissues
Kidney, skin, liver, and lungs of mice were collected for quantitative PCR evaluation of senescence markers CD26, IL-1α, p16, and p21 in tissues after treatment with TGFRt15-TGFRs or PBS control groups. 76 week old C57BL/6 aged mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment for one week prior to performing any studies. Mice were divided into two groups as follows: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). Mice were treated subcutaneously on days 0 and 10 with either PBS or TGFRt15-TGFRs at a dosage of 3 mg/kg. Seven days after the second dose of study treatment, mice were euthanized and kidneys, skin, liver, and lungs were collected and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. The samples were homogenized using a mortar and pestle in liquid nitrogen. Homogenized tissue was transferred to a new Eppendorf tube containing 1 mL of Trizol (Thermo Fischer). Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions and 1 μg of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in expression levels. Expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct _target −Ct _18S .

As shown in Figures 198-201, p21 ^CIP1 decreased in liver (Figure 198), lung (Figure 201) and skin (Figure 200) of TGFRt15-TGFRs treated mice, although there was no difference in the senescence markers CD26 and IL-1α. ) showed a decrease in expression. In the kidney (FIG. 199), both p21 ^CIP1 and IL1α markers were significantly decreased in aged mice 7 days after the second dose of TGFRt15-TGFRs.

Example 84: β-Gal Staining of Kidney Tissue by Histology Mouse kidneys were prepared to assess the senescence marker β-gal in kidney tissue after treatment with TGFRt15-TGFRs. 76-week-old C57BL/6 aged mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups as follows: PBS control group (n=6) and TGFRt15-TGFRs group (n=6). On days 0 and 10, mice were treated subcutaneously with PBS or TGFRt15-TGFRs at a dosage of 3 mg/kg. Seven days after the second dose of study treatment, mice were euthanized, kidneys were harvested and half of the kidney tissue was embedded in tissue tech cyromold containing OCT compound. Tissue tech cyromolds containing tissue were immediately frozen in the vapor phase of liquid nitrogen. Samples were further processed to cut 4-8 um thick cryostat sections (Lecia Cm1800 Cryostat) and mounted on Superfrost Plus slides. Slides containing sections were processed for aged β-galactosidase staining kit (Cell Signaling) according to the manufacturer's protocol. Tissue sections were observed under a microscope.

As shown in Figure 202, a decrease in senescence-associated β-gal ⁺ cell numbers was observed in TGFRt15-TGFRs treated mice compared to control mice (n=3). These results demonstrate that TGFRt15-TGFRs treatment can reduce senescence-associated β-gal in tissues of aged mice.

Example 85 Generation of TGFRt15*-TGFRs Fusion Proteins A fusion protein complex consisting of a TGFR/IL15RαSu fusion protein and a TGFR/TF/IL-15D8N fusion protein was generated (FIGS. 209 and 210). Human TGF-β receptor (TGFR), IL-15 alpha receptor sushi domain (IL15RaSu), tissue factor (TF), and IL-15 containing D8N variant (IL15D8N) sequences were obtained from the GenBank website. , DNA fragments of these sequences were synthesized by Genewiz. Specifically, a construct was made by joining the TGFR sequence to the N-terminal coding region of IL15RaSu and the N-terminal coding region of tissue factor 219, followed by the N-terminal coding region of IL-15D8N.

The nucleic acid sequence (including signal peptide sequence) of the TGFR/IL15RaSu construct is as follows.
(signal peptide)
ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTCTCCAGCGCCTACTCC
(single-chain human TGF-β receptor II homodimer)
ATCCCCCCCCCATGTGCAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAA GAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGAACCTTCTTTATGTGTTCCTGTAGCAG CGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCC CCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCCACGATCCCCAAGCTGCCCTAC CACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(Sushi domain of IL15 receptor α chain)
ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCT CTTTAAAGTGCATCCGG

The nucleic acid sequence (including signal peptide sequence) of the TGFR/TF/IL15D8N construct is as follows.
(signal peptide)
ATGGGAGTGAAAGTTCTTTTTGCCCTTTATTGTATTGCTGTGGCCCGAGGCC
(single-chain human TGF-β receptor II homodimer)
ATCCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAA GTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGAT GAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCCAGCT GTGCAAATTCTGCGATGTGGAGGTTTTCCACCTGCGCACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAAACCGTCTGCCACGATCCCCAAGCTGCCCTACCACGAT TTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAGAAGCCTGGCGAGACCTTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCCGAC
(human tissue factor 219)
TCAGGCACTACAAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCA CCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCTCTCGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGT GGGAACAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAACAGCCAAAACAAAACACTAATGAGTTTTTGATT GATGTGGATAAAGGAGAAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCCGGTAGAGTGTATGGGCCAGGAGAAAAGGGGGAATTCAGAGAA
(human IL-15D8N)
AACTGGGTGAATGTAATAAGTAATTTGAAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCA AGTATTCATGATACAGTAGAAATCTGATCATCCTAGCAAACAACAGTTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT

The amino acid sequence (including signal peptide sequence) of the TGFR/IL15RaSu fusion protein is as follows.
(signal peptide)
MKWVT FISLLFLFS SAYS
(single-chain human TGF-β receptor II homodimer)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(Human IL-15 receptor α sushi domain)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

The amino acid sequence (including signal peptide sequence) of the TGFR/TF/IL15D8N fusion protein is as follows.
(signal peptide)
MGVKVLFALICIAVAEA
(single-chain human TGF-β receptor II homodimer)
IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNCSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSSSDECNDNIIFSEEYNTSNPDGGGGGSGGGGGSGGGGSIPPHV QKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMNSITSICEKPQEVCVAVWRKNDINITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD
(tissue factor)
SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTF LSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE
(IL-15D8N)
NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEEELEEKNIKEFLQSFVHIVQMFINTS

The TGFR/IL15RαSu and TGFR/TF/IL-15D8N constructs were cloned into the modified retroviral expression vectors previously described (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 these two constructs in CHO-K1 cells allowed the formation and secretion of soluble TGFR/IL15RαSu-TGFR/TF/IL-15D8N protein complexes (termed TGFRt15*-TGFRs), which It can be purified by anti-TF antibody affinity.

Example 86: Binding Activity of TGFRt15-TGFRs and TGFRt15*-TGFRs to TGF-β1 and LAP The binding activity of TGFRt15-TGFRs to TGF-β1 and LAP was determined by ELISA. TGFRt15-TGFRs (5 mg/mL) were used to capture titrated TGF-β1 (TGFβ1, labeled as BioLegend) and potentially related peptides of TGF-β1 (LAP, R&D Systems). TGF-β1 was detected by biotinylated anti-TGF-β1 (0.2 mg/mL, R&D Systems) and LAP was detected by biotinylated anti-LAP (0.2 mg/mL, R&D Systems) followed by peroxidase-conjugated streptavidin (Jackson ImmunoResearch). Lab). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Surmodics IVD) was used as substrate and measured with a plate reader. As shown in FIG. 211A, the results demonstrate that TGFRt15-TGFRs bind TGF-β1 and LAP similarly and more strongly than Fc fusions.

The binding activity of TGF-β1 receptor/Fc fusions to TGF-β1 and LAP was determined by ELISA. A commercially available TGF-β1 receptor II-Fc fusion (TGFRII/Fc) was used to compare the binding activity of TGFRt15-TGFRs to TGF-β1 and LAP. TGFRII/Fc (5 mg/mL, R&D Systems) was used to capture titrated TGF-β1 and LAP. Other procedures were the same as described above. As shown in FIG. 211B, the results demonstrate that TGFRII/Fc binds to TGF-β1 and LAP similarly, with binding comparable to TGFRt15-TGFRs and stronger than Fc fusion.

Binding Activity of TGFRt15-TGFRs and TGFRt15*-TGFRs to TGF-β1 and LAP TGFRt15-TGFRs and TGFRt15*-TGFRs (10 mg/mL) were used to capture titrated TGF-β1 LAP. Other procedures were the same as described above. As shown in Figures 211C and D, the results demonstrate that TGFRt15*-TGFRs bind to TGF-β1 and LAP similarly, with binding comparable to TGFRt15-TGFRs and stronger than Fc fusions. .

Binding of TGFRt15-TGFRs and TGFRt15*-TGFRs to CTLL-2 Cells IL-2-dependent CTLL-2 cells were treated with TGFRt15-TGFRs (50 nM), 7-TF-IL15 and IL-21-IL-15RaSu) (as control fusion molecules without TGF-β1 receptor II), and PBS (as negative control) for 60 min, biotinylated secondary staining antibody (anti TF: anti-human tissue factor, HCW Biologics and anti-TGFR: anti-TGF-β receptor II: R&D Systems) followed by staining with R-phycoerythrin-streptavidin (Jackson ImmunoResearch Lab). Mean fluorescence intensity (MFI) of staining was measured by flow cytometry. As shown in FIG. 211E, the results show that TGFRt15-TGFRs bind CTLL-2 cells significantly better than other molecules, and TGFRt15*-TGFRs are less than TGFRt15-TGFRs due to IL-15 variants. indicates However, binding of 7t15-21s to CTLL-2 cells was detectable with anti-TF but not with anti-TGFR.

Example 87: Biological activity of TGFRt15-TGFRs and TGFRt15*-TGFRs by cell-based assay TGF -β1 blocking activity of TGFRt15-TGFRs and TGFRt15*-TGFRs HEK-Blue TGF-β cells (InvivoGen) were Incubated in IMDM-10 with titrated TGFRt15-TGFRs, TGFRt15*-TGFRs, and TGFRII/Fc as a control in the presence of β1 (0.1 nM, BioLegend). TGFRII/Fc is a commercially available TGF-β1 receptor II-Fc fusion (R&D Systems). After 24 hours of incubation, culture supernatants were mixed with QUANTI-Blue (InvivoGen) and incubated for 1-3 hours. OD620 values were measured with a plate reader. As shown in Figure 212A, TGFRt15-TGFRs and TGFRt15*-TGFRs had the same TGF-β1 blocking activity. In contrast, TGFRII/Fc (IC50 = 470.2 pM) has approximately 10-fold lower TGF-β1 blocking activity than TGFRt15-TGFRs (IC50 = 43.2 pM) or TGFRt15*-TGFRs (45.2 pM). was Blocking activity was calculated with GraphPad Prism 7.04.

IL-15 Activity of TGFRt15-TGFRs and TGFRt15*-TGFRs IL-15 dependent 32Dβ cells were cultured in IMDM-10 with titrated TGFRt15-TGFRs, TGFRt15*-TGFRs and IL15 as a control. Two days later WST-1 (Fisher Scientific) was added and OD450 values were measured with a plate reader. As shown in Figure 212B, TGFRt15-TGFRs (EC50 = 1641 pM) had approximately 20-fold lower IL-15 bioactivity than IL-15 itself (IC50 = 81.8 pM). As expected, TGFRt15*-TGFRs had no detectable IL-15 activity. IL-15 activity was calculated with GraphPad Prism 7.04.

Reversal of TGF-β Proliferation Suppression of CTLL-2 by TGFRt15*-TGFRs TGF-β comprises three isoforms (TGF-β1, TGF-β2 and TGF-β3) with similar biological functions. In this study, CTLL-2 cells were used to compare the biological blocking activity of TGFRt15*-TGFRs. TGFRt15*-TGFRs are structurally very similar to TGFRt15-TGFRs, but cannot be used for the IL-15 activity of TGFRt15-TGFRs. CTLL-2 cells were treated with titrated murine IL-4 (Biolegend), TGF-β (5 ng/ml, TGF-β1 (Biolegend), TGF-β2, β3 (R&D Systems)) and TGFRt15*TGFRs (21 nM, TGFRt15). *—The cells were cultured in RPMI-10 containing TGFRs:TGF-β (molar ratio=100:1) for 5 days. Cell proliferation (OD _570-600 values) was measured with a plate reader after addition of PrestoBlue (Fisher Scientific) on the last day of culture. Figure 212C shows that all three TGF-βs similarly inhibited IL-4-induced CTLL-2 proliferation in the absence of TGFRt15*-TGFRs. FIG. 212D shows that TGFRt15*-TGFRs (21 nM, molar ratio of TGF-β:TGFRt15*-TGFRs=1:100) significantly inhibited TGF-β1 and TGF-β3 of IL-4-induced CTLL-2 cell proliferation. In contrast, TGFRt15*-TGFRs had minimal reversal TGF-β2 inhibitory activity.

Example 88: Stability of TGFRt15-TGFRs Stability of TGFRt15-TGFRs by ELISA TGFRt15-TGFRs were preincubated in RPMI medium containing 50% human serum at 4°C, room temperature (RT) or 37°C for 10 days. The IL-15 and TGFβRII domains of TGFRt15-TGFRs were assessed by ELISA. Anti-TF antibody (HCW Biologics) was used to capture TGFRt15-TGFRs molecules, biotinylated anti-IL-15 (R&D Systems) was used to detect the IL-15 domain, and biotinylated anti-TGFβRII (R&D Systems) was detected. was used to detect the TGFβRII domain. A biotinylated detection antibody was probed with peroxidase-streptavidin (Jackson ImmunoResearch Lab). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Surmodics IVD) was used as substrate and OD405 values were measured with a plate reader. As shown in Figures 213A and B, the results indicate that there were no significant changes in the domains of TGFRt15-TGFRs after 10 days of incubation at 4°C, RT, or 37°C. These findings demonstrate that the IL-15 and TGFβRII domains of TGFRt15-TGFRs remain intact when incubated with human serum under the conditions evaluated.

Stability of biological activity of TGFRt15-TGFRs by cell-based assay TGFRt15-TGFRs were pre-incubated with RPMI-10 containing 50% human serum at 4°C, room temperature (RT) or 37°C for 10 days. The TGF-β1 neutralizing activity of TGFRt15-TGFRs was accessed using HEK-Blue TGF-β cells (TGF-β1 activity reporting cell line, InvivoGen). HEK-Blue TGF-β cells were incubated in IMDM-10 containing titrated TGFRt15-TGFRs in the presence of TGF-β1 (0.1 nM). After 24 hours of incubation, culture supernatants were mixed with QUANTI-Blue (InvivoGen) and incubated for 1-3 hours. OD620 values were measured with a plate reader. As shown in Figure 213C, the results indicate that there was no change in the TGF-β1 neutralizing activity of TGFRt15-TGFRs after incubation in human serum for 10 days at 4°C, RT, or 37°C. IL-15 activity of TGFRt15-TGFRs was assessed in IL-15 dependent 32Dβ cells. 32Dβ cells were cultured in IMDM-10 with titrated TGFRt15-TGFRs. Two days later WST-1 (InvitroGen) was added and OD450 values were measured with a plate reader. As shown in Figure 213D, the results indicate that there was no change in IL-15 activity of TGFRt15-TGFRs after incubation in human serum for 10 days at 4°C, RT, or 37°C.

Example 89: Reversal of TGF-β1 Immunosuppression on Human NK Cells and PBMC by TGFRt15-TGFRs and TGFRt15*-TGFRs Purified from buffy coats (4 donors, 1 blood), PBMCs were isolated from blood buffy coats (6 donors) by Ficoll-Paque (Sigma-Aldrich) density centrifugation. NK cells and PBMCs were treated with IL-15 (10 ng/mL, PeproTech) and/or TGF-β1 (10 ng/mL, Biolegend), TGFRt15-TGFRs (42 nM or 4.2 nM) or TGFRt15*-TGFRs (42 nM or 4.2 nM). 2 nM) in RPMI-10 for 3 days. Cultures were harvested and used in the following assays: cell-mediated cytotoxicity assays (Figures 214A and B) and intracellular granzyme B (Figures 214C and D) and interferon gamma (IFNγ, Figures 214E and F) flow cytometry. metric analysis.

Cultured NK cells and PBMC were used as effector cells and K562 tumor cells (ATCC) were used as target cells for cell-mediated cytotoxicity assays. A mixture of effector cells and K562 tumor cells was incubated in RPMI-10 at 37° C. for 4 hours at an E:T ratio of 4:1 for NK cells (FIG. 214A) and 20:1 for PBMCs (FIG. 214B). ). The level of dead K562 cells was determined by flow cytometry. As shown in FIGS. 214A and B, the results show that significantly fewer K562 target cells died in the presence of TGF-β1 than observed in medium control cultures, suggesting that TGF-β1 kills immune cells. It has been shown to inhibit cytotoxicity. However, in the presence of TGF-β1 and TGFRt15-TGFRs or TGFRt15*-TGFRs, there were significantly more dead K562 target cells than when cultured in TGF-β1-only conditions. These findings demonstrate that TGFRt15-TGFRs and TGFRt15*-TGFRs significantly reduced TGF-β1 immunosuppression and enhanced human NK cell and PBMC cytotoxicity against K562 target cells in a concentration-dependent manner. there is Furthermore, the IL-15 activity of TGFRt15-TGFRs further enhances human NK cell and PBMC cytotoxicity compared to that of TGFRt15*-TGFRs.

Cultured NK cells and PBMCs were stained with fluorochrome-labeled anti-CD56 and anti-CD16 human NK cell surface markers followed by fluorochrome-labeled granzyme B and IFNγ intracellular molecules (BioLegend). Granzyme B and IFNγ expression (MFI: mean fluorescence intensity) in purified NK cells and gated NK cells (CD56 ⁺ and/or CD16 ⁺ ) of PBMC cultures were analyzed by flow cytometry. As shown in Figures 214C and D, granzyme B (Figures 214C and 214D) and IFNγ (Figures 214E and 214F) expression in NK cells cultured in the presence of TGF-β1 was higher than that in cells cultured in medium alone. Significantly less than observed, indicating that TGF-β1 inhibits immune cell activation. However, in the presence of TGF-β1 and TGFRt15-TGFRs or TGFRt15*-TGFRs, there were significantly higher granzyme B and IFNγ expressing NK cell cultures than observed in cells cultured with TGF-β1 alone. TGFRt15*-TGFRs had minimal effects on granzyme B and IFNγ expression at 4.2 nM concentration. These findings demonstrate that TGFRt15-TGFRs and TGFRt15*-TGFRs significantly enhanced granzyme B and IFNγ expression in human NK cells in a concentration-dependent manner through activation of IL-15 and TGFβRII domains. .

Example 90 Half-Life of TGFRt15-TGFRs in C57BL/6 Mice The pharmacokinetics (half-life, t1/2) of TGFRt15-TGFRs were evaluated in female C57BL/6 mice. Mice were treated subcutaneously with TGFRt15-TGFRs at a dosage of 3 mg/kg. Mouse blood was collected from the tail vein at various time points and serum was prepared. TGFRt15-TGFRs concentration in mouse serum was determined by ELISA. Anti-TF antibody (anti-human tissue factor antibody generated at HCW Biologics) was used to capture TGFRt15-TGFRs molecules and biotinylated anti-TGFβRII (R&D Systems) was used to detect TGFβRII domains. A biotinylated detection antibody was probed with peroxidase-streptavidin (Jackson ImmunoResearch Lab). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Surmodics IVD) was used as substrate and OD405 values were measured with a plate reader. As shown in Figure 215, the half-life of TGFRt15-TGFR calculated with GraphPad Prism 7.04 was 18.22 hours in C57BL/6 mice.

Example 91 Toxicity of TGFRt15-TGFRs in C57BL/6 Mice A single dose of TGFRt15-TGFRs (50-400 mg/kg) was injected subcutaneously into C57BL/6 female mice (7 weeks old, n=4). Mice were weighed as shown in Figure 216 and clinical signs (mortality, morbidity, frilly hair, hunched posture, lethargy, etc.) were assessed during the experimental period. Mice administered 200 mg/kg or 400 mg/kg of TGFRt15-TGFRs exhibited decreased activity 6-8 days after treatment and no other significant clinical signs. TGFRt15-TGFRs at 200 mg/kg or 400 mg/kg caused weight loss in mice compared to the PBS group, especially on day 7 after treatment (p<0.05). Affected mice gradually recovered without dying or becoming ill after 10 days. As shown in Figure 216, these findings indicate that C57BL/6 mice can tolerate a single dose of TGFRt15-TGFRs at up to 100 mg/kg.

Example 92: Antitumor Activity of TGFRt15-TGFRs in C57BL/6 Mouse Melanoma Model Mouse B16F10 melanoma cells were injected subcutaneously into C57BL/6 mice (Jackson Laboratory) to establish a mouse melanoma model. Four days after tumor cell injection, mice were divided into different groups and received the following immunotherapies. Group 1: PBS vehicle control, Group 2: anti-tumor antibody TA99 (10 mg/kg) only control, Group 3: TA99 in combination with IL-15SA (0.05 mg/kg), Group 4: TA99 with TGFRt15-TGFRs ( 4.93 mg/kg, equivalent IL-15 activity of IL-15SA at 0.05 mg/kg), Group 5: TA99 and TGFRt15*-TGFRs (4.93 mg/kg; IL-15 activity without IL-15 activity); -15D8N mutant). Tumor volume was measured and calculated using the formula length x width x width/2. As shown in FIG. 217, mice administered anti-tumor antibody TA99 in combination with TGFRt15-TGFRs or IL15SA were significantly more stable after tumor inoculation compared to PBS, TA99 antibody alone, and TA99 in combination with TGFRt15*-TGFRs. Results showed that they had significantly smaller tumors on day 11 (p<0.05). There were no significant differences between Groups 1, 2, and 5 and between Groups 3 and 4. These findings demonstrated that the IL-15 activity of TGFRt15-TGFRs is important for the anti-tumor activity of TGFRt15-TGFRs.

Example 93: Model of Pulmonary Fibrosis - Treatment with TGFRt15-TGFRs Inflammatory and fibrotic lung disease (including idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cystic fibrosis) is a leading cause of death, Treatment options are limited. In addition, various treatments have the side effect of lung damage leading to pulmonary fibrosis. For example, pulmonary toxicity occurs in about 10% of cancer patients receiving bleomycin chemotherapy. These effects have led to the use of bleomycin treatment in rodents to model pulmonary fibrosis for the study of mechanisms involved in fibrogenesis and evaluation of potential therapeutics. To evaluate the activity of TGFRt15-TGFRs in this model, 9-week-old C57B16/j male mice were administered 50 μL of bleomycin (2.5 mg/kg, single dose) by oropharyngeal route. Seventeen days after bleomycin treatment, mice were subcutaneously administered TGFRt15-TGFRs (3 mg/kg). Mice were sacrificed 28 days after bleomycin administration. Lungs were isolated, left lungs were homogenized, and 100 μL of homogenate was assayed for hydroxyproline content as a measure of collagen deposition using a commercial kit according to the manufacturer's instructions. Data are expressed as μg of hydroxyproline content per gram of lung. As shown in Figure 218, the results indicate that TGFRt15-TGFRs therapy significantly reduced collagen deposition (ie, fibrosis) in the lungs of bleomycin-treated mice.

Example 94: In vivo characterization of the activity of TGFRt15-TGFRs and TGFRt15*-TGFRs It has been shown that protection from obesity and diabetes in leptin-deficient ob/ob mice can be achieved 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, a leptin receptor-deficient db/db mouse strain (BKS.Cg Dock7m ^+/+ Leprdb/J) were used in the study. Six-week-old db/db mice were divided into three groups (N=8 per group). Mice were injected subcutaneously with TGFRt15-TGFRs, TGFRt15*-TGFRs, or PBS at 3 mg/kg. After fasting the mice for 20 hours, blood was collected from the submandibular vein 4 days after injection. Fasting blood glucose was measured with a OneTouch UltraMini meter immediately after blood collection. As shown in Figure 219, both TGFRt15-TGFRs and TGFRt15*-TGFRs can significantly reduce fasting plasma glucose levels.

Plasma TGFβ1-3 levels were assessed to identify the cause of the treatment-related reduction in fasting plasma glucose in db/db mice. After 4 days of treatment, plasma was isolated and 30 μL of plasma was sent to EVE Technologies (Calgary, AB Canada) to assess TGFβ1-3 levels by the TGF-β3-Plex (TGFB1-3) assay. As shown in FIGS. 220A-C, both TGFRt15-TGFRs and TGFRt15*-TGFRs completely depleted plasma TGFβ1 (FIG. 220A), partially reduced TGFβ2 (FIG. 220B), and had no effect on TGFβ3. No (Figure 220C).

Lymphocyte subsets were evaluated to identify the cause of the treatment-related reduction in fasting plasma glucose in db/db mice. Four days after treatment, whole blood cells (50 μl) were treated with ACK (ammonium chloride-potassium) lysis buffer to lyse red blood cells. Lymphocytes were then stained with PE-Cy7-anti-CD3, BV605-anti-CD45, PerCP-Cy5.5-anti-CD8a, BV510-anti-CD4, and APC-anti-NKp46 (all antibodies from BioLegend). to assess T cell and NK cell populations. Cells were further permeabilized, fixed with eBioscience Foxp3/Transcription Factor Staining Buffer Set (Cat #00-5523-00, ThermoFisher) and AF700- in eBioscience Permeabilization Buffer (Cat #00-8333-56, ThermoFisher). Staining with anti-Ki67 and FITC-anti-granzyme B was used to assess proliferation and activation of T cells and NK cells. Another set of lymphocytes was first stained with PE-Cy7-anti-CD3, BV605-anti-CD45, BV510-anti-CD4, and apc-Cy7-anti-CD25, then permeabilized and analyzed by eBioscience Foxp3/ Treg were stained with PE-anti-Foxp3 in eBioscience Permeabilization Buffer (Cat. #00-8333-56, ThermoFisher) and fixed with Transcription Factor Staining Buffer Set (Cat. #00-5523-00, ThermoFisher). Cell populations were evaluated.

TGFRt15-TGFRs increased populations of NK cells (Figure 221A) and CD8 ⁺ T cells (Figure 221D), stimulated proliferation of NK cells (Figure 221B) and CD8 ⁺ T cells (Figure 221E), Figure 221C) was activated. TGFRt15*-TGFRs had no effect on either cell population (FIGS. 221A-E). Both TGFRt15-TGFRs and TGFRt15*-TGFRs had no effect on CD4 ⁺ T cells, CD19 ⁺ B cells and CD4 ⁺ CD25 ⁺ Foxp3 ⁺ Treg cells.

In conclusion, both TGFRt15-TGFRs and TGFRt15*-TGFRs reduced fasting plasma glucose levels and both TGFRt15-TGFRs and TGFRt15*-TGFRs completely depleted plasma TGFβ1 in db/db mice. However, only TGFRt15-TGFRs activated NK cells and enhanced proliferation of CD8 ⁺ T cells and NK cells. Based on these results, depletion of TGFβ1 is likely involved in reducing fasting plasma glucose, and blockade of TGF-β/Smad3 signaling has a role in preventing obesity and diabetes in ob/ob mice. Show fulfillment.

Example 95 In Vitro Characterization of Activity of TGFRt15-TGFRs and TGFRt15*-TGFRs TGFRII was demonstrated to interact with TGFβ1-3. There are no literature reports showing interactions between TGFRII and potential TGFβ. To assess whether TGFRt15-TGFRs, TGFRt15*-TGFRs, and TGFRII-Fc interact with cryptic TGFβ, a 2.5 nM human cryptic TGFβ1-his tag (catalog number TG1-H524x, Acro Biosystems) was used. Alternatively, the control protein CD39-his tag (Lot #58-49/51, HCW Biologics) was dissolved in 50 mM carbonate buffer pH 9.4 (100 μl/well) and plated on an ELISA plate (Cat #80040LE0910, ThermoFisher) at 4°C. Coated overnight. The next day, the plates were washed three times with ELISA wash buffer (phosphate buffered saline containing 0.05% Tween 20) and the plates were blocked with blocking buffer (1% BSA-PBS) for 1 hour before Decreasing concentrations of TGFRt15-TGFRs, TGFRt15*-TGFRs, or TGFRII-Fc in 0.09 nM blocking buffer were added to the plates and the plates were incubated at 25° C. for 1 hour. Plates were washed 3 times with ELISA wash buffer. A biotinylated anti-TGFRII antibody (catalog number BAF241, R&D Systems) at 0.1 μg/mL detection antibody was added to the plate and incubated at 25° C. for 1 hour. Plates were washed and 0.25 μg/mL horseradish peroxidase-streptavidin (Code No. 016-030-084, Jackson ImmunoResearch) was added to the plates and incubated at 25° C. for 30 minutes. Plates were washed and HRP substrate ABTS (catalog number ABTS-1000-01, Surmodics) was added to the plates and incubated at 25° C. for 20 minutes. Plates were read at OD405 nm using a microplate reader (Multiscan Sky, Thermo Scientific). As shown in FIG. 222A, both TGFRt15-TGFRs and TGFRt15*-TGFRs interacted similarly with potential TGFβ1. However, TGFRII-Fc interacted with cryptic TGFβ1 with lower affinity than that seen with TGFRt15*-TGFRs (FIG. 222B). The results show that TGFRt15-TGFRs, TGFRt15*-TGFRs, and TGFRII-Fc interact with potential TGFβ1, with TGFRt15-TGFRs, TGFRt15*-TGFRs surprisingly exhibiting higher affinity interactions than TGFRII-Fc. We proved that.

Example 96: Prothrombin Time Test The Prothrombin Time (PT) test is designed to measure the time it takes plasma to clot after mixing with tissue factor and an optimal concentration of calcium. A mixture of tissue factor with phospholipids (called thrombinplastin) acts as an enzyme that converts prothrombin to thrombin and fibrinogen to fibrin, thereby causing blood clotting. Innovin is a lipidated recombinant human TF243 and is used as an experimental standard. In the PT assay, a shorter PT time (clotting time) indicates higher TF-dependent coagulation activity, and a longer PT (clotting time) means lower TF-dependent coagulation activity.

Briefly, 0.1 mL of normal human plasma (Ci-Trol Coagulation Control, Level I) was preheated to 37° C. for 3 minutes. Test samples (TGFRt15-TGFRs) diluted in 0.2 mL of various dilutions of Innovin or PT assay buffer (50 mM Tris-HCl, pH 7.5, 14.6 mM CaCl ₂ , 0.1% BSA) were added to plasma. Plasma clotting reaction was initiated by addition. Clotting times were monitored and reported by a START PT Analyzer (Diagnostica Stago, Parsippany, NJ.).

As seen in Figure 223, different amounts of Innovin added to the PT assay (Innovin reconstituted in purified water equivalent to 10 nM lipidated recombinant human TF243 is considered to be 100% Innovin) , indeed showed an inverse correlation between the amount of TF243 added in the PT assay and PT time. For example, 1% Innovin had a PT time of approximately 25.0 seconds, while 100% Innovin had a PT time of 8.5 seconds.

Figure 224 shows the results of the PT study of TGFRt15-TGFRs. In contrast to Innovin, TGFRt15-TGFRs exhibit prolonged PT times nearly identical to buffer, indicating very low or no clotting activity.

The coagulant effect of TGFRt15-TGFRs in the presence of CTLL cells was also evaluated. Binding experiments performed confirmed that TGFRt15-TGFRs can bind to CTLL cells. The TGFRt15-TGFRs clotting assay in the presence of CTLL cells more closely reflects their potent clotting activity in vivo. TGFRt15-TGFRs were pre-incubated with CTLL cells at 37° C. for 20-30 minutes in PT assay buffer. The PT assay was then proceeded as described above. Figure 224 shows that the mixture of TGFRt15-TGFRs and CTLL cells had a slightly shorter clotting time (154.6 seconds) than TGFRt15-TGFRs alone (167.6 seconds) or CTLL cells alone (161.9 seconds). indicate. However, the clotting time of 154.6 seconds is still significantly longer than the Innovin clotting time of 8.5 seconds.

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 in the presence of cells capable of binding to TGFRt15-TGFRs. .

Example 97: Gene Expression of Senescence Markers in Tissues of Young and Aged Mice After Treatment with TGFRt15-TGFRs or PBS and Short-Term (10 Days) or Long-Term (60 Days) Follow-up Purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups and treated subcutaneously with either PBS (PBS control group) or TGFRt15-TGFRs at a dosage of 3 mg/kg (TGFRt15-TGFRs group). Mice were euthanized either 10 or 60 days after treatment and kidneys were harvested for quantitative PCR assessment of expression levels of senescence markers PAI1, IL-1α, IL6, and TNFα. Harvested kidneys were stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were homogenized with 1 mL of Trizol (Thermo Fischer) using a homogenizer. The homogenized tissue was transferred to a new Eppendorf tube. Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using QuantiTect reverse transcription (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in expression levels. The expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct target−Ct18S. Untreated 6-week-old mice (young) were used as controls to compare gene expression levels with aged mice.

As shown in FIG. 225, the results show that gene expression of PAI-1, IL-1α, IL6, and IL-1β in the kidney increased as mice age, as expected with an age-dependent increase in cellular senescence. It shows that it increased with Treatment of 72-month-old mice with a single dose of TGFRt15-TGFRs resulted in a significant and long-lasting effect on decreased gene expression of senescence markers in the kidneys and treatment of spontaneous senescent cells in the kidneys of aged mice. suggesting an associated decrease.

As shown in Figure 226, the results show that treatment of 72-month-old mice with a single dose of TGFRt15-TGFRs mediated significant and long-term effects in reducing IL-1α and IL6 gene expression in the liver. , suggesting a treatment-related reduction of spontaneous senescent cells in the liver of aged mice.

72 week old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into two groups and treated subcutaneously with either PBS (PBS control group) or TGFRt15-TGFRs at a dosage of 3 mg/kg (TGFRt15-TGFRs group). Mice were euthanized either 10 or 60 days after treatment and kidneys were harvested for assessment of protein levels of the senescence marker PAI-1 by tissue ELISA. Harvested kidneys were stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were homogenized with 0.3 mL of extraction buffer (Abcam) using a homogenizer. The homogenized tissue was transferred to a new Eppendorf tube. Protein levels in homogenized tissues were quantified using the BCA Protein assay kit (Pierce). Mouse PAI-1 ELISA (R&D System) was performed using 200 mg of tissue homogenate. Concentrations of PAI-1 were calculated as picograms per milligram of tissue based on the standard curve.

As shown in FIG. 227, protein levels of the senescence marker PAI-1 were decreased in the kidneys of TGFRt15-TGFRs-treated aged mice compared to the PBS group 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 indicate that a single treatment of TGFRt15-TGFRs had a significant and long-lasting effect on the reduction of spontaneous senescent cells in tissues of aged mice (senescence marker genes and proteins (measured by reduced expression).

Example 98: Comparison of TGFRt15-TGFRs and TGFRt15*-TGFRs (IL-15 Mutants) Treatment in Reducing Gene Expression of Senescence Markers in Aged Mice Tissues 72-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into five groups as follows: saline control group (n = 8), TGFRt15-TGFRs group (n = 8), IL15SA group (n = 8), TGFRt15*-TGFRs group (n = 8), and the IL15SA+TGFRt15*-TGFRs group (n=8). Mice were given PBS, TGFRt15-TGFRs (3 mg/kg), TGFRt15*-TGFRs (3 mg/kg), IL15SA (0.5 mg/kg), or TGFRt15*-TGFRs (3 mg/kg) + IL15SA (0.5 mg/kg). ) was administered subcutaneously. Mouse blood was prepared to evaluate different subsets of immune cells after treatment with TGFRt15-TGFRs and other agents. Mouse blood was collected on day 17 from the submandibular vein into tubes containing EDTA. Whole blood was centrifuged at 3000 RPM for 10 minutes in a microcentrifuge to collect plasma. Plasma was stored at −80° C. and whole blood was processed for immune cell phenotyping by flow cytometry. Whole blood RBCs were lysed in ACK buffer for 5 minutes at room temperature. The remaining cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells in the fluid, cells were stained with antibodies specific for cell surface CD3, CD45, CD8 and NK1.1 (BioLegend) for 30 min at room temperature (RT). After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figure 228, the results indicate that aged mice were treated with TGFRt15-TGFRs. IL15SA (positive control) or TGFRt15*-TGFRs+IL15SA mediated an increase in the percentage of CD3 ⁺ CD8 ⁺ , CD3 ⁻ NK1.1 ⁺ , and CD3 ⁺ CD45 ⁺ immune cells in the blood, whereas treatment with TGFRt15*-TGFR had little or no effect on the percentages of these cell populations. These results suggest that IL-15 activity of TGFRt15-TGFRs plays a role in increasing CD8 ⁺ T cells and NK cells in the blood of aged mice.

As shown in Figure 229, the results indicate that aged mice were treated with TGFRt15-TGFRs. IL15SA (positive control) or TGFRt15*-TGFRs+IL15SA mediated an increase in the percentage of splenic CD3 ⁺ CD8 ⁺ , CD3 ⁻ NK1.1 ⁺ , and CD3 ⁺ CD45 ⁺ immune cells, whereas treatment with TGFRt15*-TGFR , had little or no effect on the percentages of these cell populations. These results suggest that IL-15 activity of TGFRt15-TGFRs plays a role in increasing splenic CD8 ⁺ T cells and NK cells in aged mice.

72 week old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into five groups as follows: saline control group (n = 8), TGFRt15-TGFRs group (n = 8), IL15SA group (n = 8), TGFRt15*-TGFRs group (n = 8), and IL15SA, including the TGFRt15*-TGFRs group (n=8). Mice were given PBS, TGFRt15-TGFRs (3 mg/kg), TGFRt15*-TGFRs (3 mg/kg), IL15SA (0.5 mg/kg), or TGFRt15*-TGFRs (3 mg/kg) + IL15SA (0.5 mg/kg). ) was administered subcutaneously. Mouse kidney, liver to assess gene expression of senescence markers p21, PAI1, IL-1α, and IL6 by quantitative PCR in tissues after treatment with TGFRt15-TGFRs, TGFRt15*-TGFRs, or control groups. , and lungs were collected. Mice were euthanized 17 days after treatment and kidneys, livers and lungs were collected and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were homogenized with 1 mL of Trizol (Thermo Fischer) using a homogenizer. The homogenized tissue was transferred to a new Eppendorf tube. Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using QuantiTect reverse transcription (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in expression levels. The expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct target−Ct18S.

As shown in Figures 230A-D, treatment of 72-month-old mice with a single dose of TGFRt15-TGFRs or TGFRt15*-TGFRs significantly reduced p21, PAI1, IL-1α, and IL6 gene expression in kidney and liver. suggesting a process associated with the reduction of spontaneous senescent cells in the kidney and liver of aged mice. The results of this study suggest that both the IL-15 and TGF-β trap activities of TGFRt15-TGFRs can reduce spontaneous senescent cells in aged mouse tissues.

Example 99: Blood of Immunophenotypic Mice After Treatment with IL-15 Based Agents : IL-15 Based Agents: TGFRt15-TGFRs, IL-15 Superagonist (IL-15SA), and IL- were prepared to assess changes in various subsets of immune cells after treatment with IL-15 fusions of D8N mutants that knocked out 15 activity (TGFRt15*-TGFRs). Six-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into groups (n=6/group) and treated with: 1) PBS (saline) control, 2) docetaxel, 3) docetaxel with TGFRt15-TGFRs, 4) docetaxel with IL15SA, 5) Docetaxel with IL-15 variants (TGFRt15*-TGFRs) and 6) IL-15 superagonist (IL-15SA) and Docetaxel with TGFRt15*-TGFRs. Three doses of docetaxel (10 mg/kg) on days 1, 4, and 7 induced senescence in mice. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs, IL-15SA, or a combination of the above. TGFRt15-TGFRs and TGFRt15*-TGFRs were administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.05 mg/kg. Mouse blood was collected into EDTA tubes from the submandibular vein on day 3 after study drug treatment. Whole blood was centrifuged at 3000 RPM for 10 minutes in a microcentrifuge to collect plasma. Plasma was stored at −80° C. and whole blood was processed for immune cell phenotyping by flow cytometry. RBCs were lysed with ACK buffer at 37° C. for 5 minutes. The remaining cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells in the blood, cells were stained with antibodies against cell surface CD4, CD45, CD19, CD8 and NK1.1 (BioLegend) for 30 min at room temperature (RT). After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). Cells were treated with permeabilization buffer (Invitrogen) for 20 minutes at 40° C. and then washed with permeabilization buffer (Invitrogen). Cells were then stained for an intracellular marker of proliferation (Ki67) for 30 minutes at room temperature. After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in FIGS. 231A and 231B, the results show that treatment of mice with TGFRt15-TGFRs, IL15SA (positive control), or TGFRt15*-TGFRs+IL15SA increased the percentage of CD8 ⁺ T cells and NK1.1 ⁺ cells in the blood and Treatment with TGFRt15*-TGFRs had little or no effect on the percentage of these cell populations, indicating that it mediated an increase in proliferation (measured by Ki67). These results suggest that the IL-15 activity of TGFRt15-TGFRs plays a role in the increase of CD8 ⁺ T cells and NK cells in the blood of aged mice after chemotherapy.

Example 100 Evaluation of Gene Expression of Senescence Markers p21 and CD26 in Lung and Liver Tissues of Mice After Chemotherapy and Treatment with IL-15 Based Agents It was later evaluated on tissues from normal mice. Six-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were divided into 6 groups and treated with: 1) PBS (saline) control (n=5), 2) docetaxel (n=8), 3) docetaxel with TGFRt15-TGFRs (n=8). 4) docetaxel with IL15SA (n=8), 5) docetaxel with IL-15 variants (TGFRt15*-TGFRs) (n=8) and 6) IL-15 superagonist (IL-15SA) with TGFRt15*. - Docetaxel with TGFRs (n=6). Three doses of docetaxel (10 mg/kg) on days 1, 4, and 7 induced senescence in mice. On day 8, mice were treated subcutaneously with PBS, TGFRt15-TGFRs, TGFRt15*-TGFRs, IL-15SA, or the following combinations. TGFRt15-TGFRs and TGFRt15*-TGFRs were administered at a dosage of 3 mg/kg and IL-15SA was administered at 0.5 mg/kg. Mouse tissues were prepared to assess differential gene expression of senescence markers. Mice were euthanized 7 days after study drug treatment and liver and lung tissues were collected and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. The samples were homogenized using a mortar and pestle in liquid nitrogen. Homogenized tissue was transferred to a new Eppendorf tube containing 1 mL of Trizol (Thermo Fischer). Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions and 1 μg of total RNA was used for cDNA synthesis using the QuantiTect reverse transcription kit (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in expression levels. The expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct target−Ct18S.

As shown in Figure 232, gene expression of the senescence markers p21 and CD26 was significantly higher in lung tissues of docetaxel-treated mice (Figure 232A) and (Fig. 232A) compared to gene expression in tissues of saline-treated mice. p21 was induced in Figure 232B), as well as in liver tissue (Figure 232C). Senescence markers p21 and CD26 in the lung and p21 gene expression in the liver by TGFRt15-TGFRs, IL-15SA, and IL-15SA in combination with the TGFRt15*-TGFRs mutant compared to chemotherapy-treated controls decreased in chemotherapy-treated mice after subsequent treatments. However, TGFRt15*-TGFRs mutant treatment was unable to affect chemotherapy-induced senescence marker gene expression in these tissues. These results indicate that IL-15 activity is important for elimination of TIS senescent cells in normal tissues of mice.

Example 101: TGFRt15-TGFRs Treatment Enhances Immune Cell Proliferation, Expansion, and Activation in the Peripheral Blood of B16F10 Tumor-Bearing Mice C57BL/6 mice were injected subcutaneously with 0.5×10 6 B16F10 cells ^. After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. Blood was collected from the submandibular vein on days 3, 5, and 10 after immunotherapy treatment (day 8). RBCs were lysed with ACK lysis buffer, lymphocytes were washed and stained with antibodies specific for cell surface expression of NK, CD8, CD25 and granzyme B (GzB) (BioLegend) for 30 min at room temperature (RT). bottom. After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). After two washes, cells were resuspended in fixation buffer. After fixation, cells were washed and treated with permeabilization buffer (Invitrogen) for 20 min at 4° C. followed by washing with permeabilization buffer (Invitrogen). Cells were then stained for an intracellular marker of proliferation (Ki67) for 30 minutes at room temperature. After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figures 233A and B, peripheral blood analysis revealed predominantly proliferating Ki67-positive NK and CD8 ⁺ cells on day 3 after TGFRt15-TGFRs + TA99 therapy when compared to saline or chemotherapy-treated groups. indicated to do. Expansion of NK and CD8 ⁺ cells was seen on days 3 and 5 after immunotherapy. Ten days after immunotherapy, NK cells were still proliferating, but CD8 ⁺ cells did not proliferate in the blood. These cells also expressed the activation markers CD25 and granzyme B after TGFRt15-TGFRs+TA99 therapy when compared to immune cells in saline or chemotherapy treated groups. These effects are consistent with the immunostimulatory activity of TGFRt15-TGFRs.

Example 102: TGFRt15-TGFRs Treatment Decreases TGFβ Levels in Plasma of B16F10 Tumor-Bearing Mice C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. Blood was collected submandibularly on days 1, 3, 5, and 10 after immunotherapy treatment in tubes containing EDTA and immediately placed on ice. Blood was centrifuged at 3,000 rpm for 15 minutes at room temperature to separate plasma. Plasma samples were aliquoted and stored at -80°C. Plasma TGFβ levels were analyzed by using the cytokine array, TGFβ3plex (TGFβ1-3) from Eve Technologies, Calgary, AL, Canada.

As shown in Figure 234, the results showed that administration of TGFRt15-TGFRs + TA99 increased TGF-β1, TGF-β2, TGF-β1, TGF-β2, and reduced plasma levels of TGF-β3. This effect is consistent with the TGF-β agonistic activity of TGFRt15-TGFRs.

Example 103: TGFRt15-TGFRs Treatment Reduces Levels of Proinflammatory Cytokines in Plasma of B16F10 Tumor-Bearing Mice C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. On days 1, 3, 5, and 10 after immunotherapy treatment (day 8), blood was collected from the submandibular vein into tubes containing EDTA and immediately placed on ice. Blood was centrifuged at 3,000 rpm for 15 minutes at room temperature to separate plasma. Plasma samples were aliquoted and stored at -80°C. Aliquots were diluted 2-fold in PBS and analyzed using the Mouse Cytokine Array Proinflammatory Focus 10 Plex (MDF10) assay.

As shown in FIG. 235, the results showed that administration of TGFRt15-TGFR+TA99 increased IL2, IL-1β, IL6, MCP-1, and GM in tumor-bearing mice 10 days after treatment when compared to the chemotherapy group. - shows reduced plasma levels of CSF. This effect is consistent with the immunostimulatory activity of TGFRt15-TGFRs.

Example 104: TGFRt15-TGFRs Treatment Enhances NK and CD8+ Expansion in the Spleens of B16F10 Tumor-Bearing Mice C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. Mice were sacrificed on days 3, 5, and 10 after immunotherapy (day 8) and spleens were collected. The spleen was crushed with the flat back end of a sterile piston/plunger of a 3cc syringe to release the splenocytes. Splenocytes were passed through a 70 μM cell strainer and homogenized into a single cell suspension. RBCs were lysed with ACK lysis buffer, splenocytes were washed and stained with antibodies for cell surface expression of NK and CD8 (BioLegend) for 30 minutes at room temperature. After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figure 236, an expansion of NK cells and CD8 ⁺ cells was seen in the spleen on days 3 and 5 after TGFRt15-TGFRs + TA99 therapy when compared to saline or chemotherapy treated groups. Levels of NK cells (but not CD8 ⁺ cells) were found to be still elevated 10 days after immunotherapy in the spleens of tumor-bearing mice when compared to those in chemotherapy group spleens. These effects are consistent with the immunostimulatory activity of TGFRt15-TGFRs.

Example 105: TGFRt15-TGFRs Treatment Enhances Glycolytic Activity of Splenocytes in B16F10 Tumor-Bearing Mice C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. Mice were sacrificed on days 3, 5, and 10 after immunotherapy (day 8) and spleens were collected. The spleen was crushed with the flat back end of a sterile piston/plunger of a 3cc syringe to release the splenocytes. Splenocytes were passed through a 70 μM cell strainer and homogenized into a single cell suspension. RBCs were lysed with ACK lysis buffer and splenocytes were washed and counted. To measure splenocyte glycolytic activity, cells were washed, resuspended in seahorse medium, and resuspended at 4×10 ⁶ cells/mL. Cells were seeded at 50 μL/well in Cell-Tak coated Seahorse Bioanalyzer XFe96 culture plates in Seahorse XF RPMI medium pH 7.4 supplemented with 2 mM L-glutamine for glycolytic stress studies. Cells were allowed to attach to the plates for 30 minutes at 37°C. Additionally, 130 μL of assay medium was added to each well of the plate (also background wells). Plates were incubated for 1 hour at 37°C, non- _CO2 incubator. For the glycolytic stress test, the calibration plate contained a 10x solution of glucose/oligomycin/2DG prepared in Seahorse assay medium and 20 μL of glucose/oligomycin/2DG was calibrated overnight. was added to each port of the extracellular flux plate. The glycolytic stress test is based on extracellular acidification rate (ECAR) and measures three key parameters of glycolytic function including glycolysis, glycolytic capacity, and glycolytic reserve. A full ECAR assay was performed in four phases: non-glycolytic acidification (no drug), glycolytic (10 mM glucose), maximal glycolytic induction/glycolytic capacity (2 μM oligomycin), and glycolytic reserve (100 mM 2-DG). consists of stages. At the end of the experiment, data were exported as Graph Pad Prism files. The XF Glycolytic Stress Test Report Generator automatically calculated the parameters of the XF Cellular Glycolytic Stress Test from the 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 therapy were significantly higher than those of saline or chemotherapy-treated groups. , enhanced basal glycolysis, capacity and reserve rate. However, no significant difference was observed in splenocyte glycolytic activity 10 days after immunotherapy. These effects are consistent with the immunostimulatory activity of TGFRt15-TGFRs.

Example 106: TGFRt15-TGFRs Treatment Enhances Splenocyte Mitochondrial Respiration in B16F10 Tumor-Bearing Mice C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. Mice were sacrificed on days 3, 5, and 10 after immunotherapy (day 8) and spleens were collected. The spleen was crushed with the flat back end of a sterile piston/plunger of a 3cc syringe to release the splenocytes. Splenocytes were passed through a 70 μM cell strainer and homogenized into a single cell suspension. RBCs were lysed with ACK lysis buffer and splenocytes were washed and counted. To measure splenocyte mitochondrial respiration, cells were washed, resuspended in seahorse medium, and resuspended at 4×10 ⁶ cells/mL. Cells were seeded at 50 μL/well in Cell-Tak coated Seahorse Bioanalyzer XFe96 culture plates in Seahorse XF RPMI medium pH 7.4 supplemented with 2 mM L-glutamine for glycolytic stress studies. For mitochondrial stress tests, cells were seeded in Seahorse XF RPMI medium, pH 7.4, supplemented with 10 mM glucose and 2 mM L-glutamine. Cells were allowed to attach to the plates for 30 minutes at 37°C. Additionally, 130 μL of assay medium was added to each well of the plate (also background wells). Plates were incubated for 1 hour at 37°C, non- _CO2 incubator. For the mitochondrial stress test, the calibration plate contained a 10x oligomycin/FCCP/rotenone solution prepared in Seahorse assay medium and 20 μL of oligomycin, FCCP, and rotenone were calibrated overnight. was added to each port of the extracellular flux plate. Oxygen consumption rate (OCR) was measured using an XFe96 Extracellular Flux Analyzer. A complete OCR analysis consisted of four stages: basal respiration (no drug), ATP-related respiration/proton leak (1.5 μM mM oligomycin), maximal respiration (2 μM FCCP), and pre-respiration (0.5 μM rotenone). rice field. At the end of the experiment, data were exported as Graph Pad Prism files. The XF Mitochondrial Stress Test Report Generator automatically calculates the parameters of the XF Mitochondrial Stress Test from Wave data exported to Excel. Data were analyzed by using Wave software (Agilent).

As shown in FIGS. 238A and B, splenocytes isolated from tumor-bearing mice on days 3 and 5 after TGFRt15-TGFR+TA99 therapy were significantly higher than those of saline or chemotherapy-treated groups. , enhanced basal respiration, mitochondrial respiration, capacity and ATP production. However, no significant difference was observed in splenocyte mitochondrial respiration 10 days after immunotherapy. These effects are consistent with the immunostimulatory activity of TGFRt15-TGFRs. Metabolic pathways such as oxidative metabolism and glycolysis are known to preferentially facilitate cell fate decisions and effector functions of immune cells. Thus, TGFRt15-TGFRs mediate increased glycolytic activity and mitochondrial respiration may be associated with activation of NK and CD8 ⁺ immune cells in mouse blood, spleen, and tumors.

Example 107: TGFRt15-TGFRs Treatment Enhances NK and CD8 Immune Cell Infiltration (TIL) into Tumors in B16F10 Tumor-Bearing Mice C57BL/6 mice were injected subcutaneously with 0.5×10 6 B16F10 ^cells . After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. Mice were sacrificed and tumors were harvested on days 3, 5, and 10 after immunotherapy. To determine tumor-infiltrating immune cells, tumor tissue was dissociated into single cell suspensions by collagenase digestion. Single cell suspensions were layered onto Ficoll-Paque medium followed by density gradient centrifugation to separate lymphocytes and tumor cells. Cells were centrifuged at 1000 g for 20 minutes at 20° C. with slow acceleration and break off. After centrifugation, Ficoll-Paque clearly separates the two layers. TILs are found at the interface between medium and Ficoll-Paque, while the pellet is composed of tumor cells. TILs were carefully removed from the interface and washed with complete RPMI medium. After washing, RBCs were lysed in ACK buffer for 5 minutes at room temperature. Cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells within the tumor, cells were stained with antibodies against cell surface CD8, NK1.1, CD25, and GzB (BioLegend) for 30 minutes at room temperature. After surface staining, the remaining cells were washed (1500 RPM, 5 minutes at room temperature). After two washes, cells were resuspended in fixation buffer. After fixation, cells were washed and treated with permeabilization buffer (Invitrogen) for 20 minutes at 4° C., followed by washing with permeabilization buffer (Invitrogen). Cells were then stained for an intracellular marker of proliferation (Ki67) for 30 minutes at room temperature. After washing twice, cells were 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 3 days after therapy. An expansion of NK and CD8 ⁺ cells (based on % lymphocytes in tumor) was seen at days 3 and 5 after TGFRt15-TGFRs+TA99 therapy when compared to chemotherapy-treated groups. Tumor CD8 ⁺ cells were elevated even 10 days after immunotherapy. Both NK and CD8 ⁺ showed expression of the activation markers CD25 and granzyme B 3 days after TGFRt15-TGFRs+TA99 therapy when compared to immune cells in the chemotherapy-treated group. These effects are consistent with the immunostimulatory activity of TGFRt15-TGFRs and parallel changes seen in blood and splenocytes of tumor-bearing mice.

Example 108: Histopathological Analysis of Tumors After TGFRt15-TGFRs Treatment C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice were dosed with doxetaxel chemotherapy (10 mg/kg) three times on days 1, 4 and 7, and a monoclonal antibody targeting the tumor antigen anti-TYRP-1 antibody TA99 (200 μg). A single dose of TGFRt15-TGFRs (3 mg/kg) was administered on day 8 in combination with Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) on days 1, 4, and 7 were used as controls. Blood was collected from the submandibular vein on days 1, 3, 5, and 10 after immunotherapy treatment (day 8). Mice were sacrificed 10 days after immunotherapy and tumors were isolated. For histological analysis, tumor samples were fixed in 10% formalin solution, embedded in paraffin and sectioned at 5 μm. Sections were stained with H&E to assess tissue and cell morphology. Sections were scored based on tumor mitotic and necrotic activity. The percentage of tumor necrosis was scored as +1 (0-20%), +2 (20-40%), and +3 (40-60%). Tumor mitotic index was scored as +1 = moderate (1-5 per high power field) and +2 = extensive (>5 per high power field).

As shown in Figure 240, tumors exhibited less mitotic and necrotic activity after TGFRt15-TGFRs + TA99 treatment. The mitotic index correlates with dividing cells and the presence of necrosis is a more aggressive feature and a measure of poor prognosis. Therefore, TGFRt15-TGFRs are promising therapies in preclinical mouse models for testing combination tumor immunotherapies.

Example 109: Anti-PD-L1 Antibody Combined with TGFRt15-TGFRs+TA99 and Chemotherapy in B16F10 Melanoma Mouse Model C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice received three doses of doxetaxel chemotherapy (10 mg/kg) on days 1, 4 and 7. Tumor-bearing mice treated with saline or doxetaxel chemotherapy (10 mg/kg) alone on days 1, 4, and 7 were used as controls. The remaining mice were randomly divided into two groups, one group treated with anti-mPD-L1 antibody (2×10 mg/kg) and the other group treated with TGFRt15-1 containing TA99 (200 μg) on day 8. treated with TGFRs (3 mg/kg). Six days later, mice that had been administered TGFRt15-TGFRs containing TA99 were administered anti-mPD-L1 antibody (2×10 mg/kg), and mice that had been administered anti-mPD-L1 antibody were treated with TGFRt15-TGFRs containing TA99 (200 μg). Treated with TGFRs (3 mg/kg). Anti-mPD-L1 antibodies were administered twice on days 8 and 10 or on days 14 and 16. Tumor growth was monitored by caliper measurements 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. N=6-8 mice/group.

As shown in FIG. 24I, administration of TGFRt15-TGFR+TA99 followed by anti-PD-L1 antibody treatment was associated with higher cytotoxicity in B16F10 tumor-bearing mice compared to treatment with anti-PD-L1 antibody followed by TGFRt15-TGFRs+TA99. Resulted in good anti-tumor activity. Therefore, the combination of TGFRt15-TGFRs and anti-PD-L1 antibodies may be advantageous in treating tumors resistant to anti-PD-L1 antibody therapy.

Example 110: The anti-tumor effect of TGFRt15-TGFRs in the B16F10 melanoma mouse model was demonstrated by treating groups of NK- and CD8+ T cell-dependent C57BL/6 mice (N=6-8 mice/group) with NK1. Three doses of 1Ab (500 μg) or CD8 ⁺ a (500 μg) antibodies were administered intraperitoneally to deplete NK and CD8 cells. Blood was collected prior to B16F10 tumor implantation and analyzed for NK and CD8 ⁺ lymphocyte levels. Untreated mice served as an immunocompetent control. C57BL/6 mice were injected subcutaneously with 0.5×10 ⁶ B16F10 cells. After tumor inoculation (day 0), mice were dosed with docetaxel (10 mg/kg) three times on days 1, 4, and 7, followed by TGFRt15-TGFRs (3 mg/kg) + TA99 (200 μg) on day 8. A single dose was administered. Tumor growth was monitored by caliper measurements 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 reduction in B16F10 tumor volume when compared to tumors in saline or chemotherapy treated groups. showed that. However, there was no effect of immunotherapy on anti-antitumor activity when NK and CD8 ⁺ cell subsets in mice were depleted. This experiment indicates that both NK and CD8 ⁺ immune cells play important roles in TGFRt15-TGFRs-mediated anti-tumor activity.

Example 111: Comparison of TGFRt15-TGFRs and TGFRt15*-TGFRs Treatment in Reducing Senescence Markers in Liver and Lung Tissues of B16F10 Tumor-Bearing Mice After Chemotherapy 6-8 week old C57BL/6 mice were purchased from Jackson Laboratory . Mice were housed in a temperature and light controlled environment. Mice were divided into five groups as follows: saline control group (n=7), docetaxel (DTX) group (n=7), DTX+TGFRt15-TGFRs group (n=7), DTX+TGFRt15*-TGFRs group. (n=7), and the DTX+IL15SA group (n=7). Mice were implanted on day 0 with B16F10 tumor cells (1×10 ⁷ cells/mouse). Mice were treated subcutaneously on days 1, 4, and 7 with 10 mg/kg docetaxel. On day 8, mice were dosed subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg), TGFRt15*-TGFRs (3 mg/kg), or IL15SA (0.5 mg/kg). Mice were euthanized on day 17 after treatment and senescence markers p21, IL-1α, and IL6 were detected for liver by quantitative PCR in tissues after treatment with TGFRt15-TGFRs, or TGFRt15*-TGFRs, and control groups. Liver and lung were harvested to assess p21 and IL-1α gene expression for lung. Harvested kidneys were stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were homogenized with 1 mL of Trizol (Thermo Fischer) using a homogenizer. The homogenized tissue was transferred to a new Eppendorf tube. Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using QuantiTect reverse transcription (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in expression levels. The expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct target−Ct18S.

As shown in FIG. 243, senescence markers p21, IL-1α, and IL6 increased significantly in tumors treated with TGFRt15-TGFRs and TGFRt15*-TGFRs when compared to gene expression in tissues of chemotherapy-treated mice. Both bearing mice showed decreased gene expression in liver (A) and lung (B) tissues.

Example 112: TGFRt15-TGFRs-treated B16F10 melanoma cells in reduction of chemotherapy-induced senescent tumor cells in vivo were stably transduced with a GFP lentiviral plasmid and tumor cells expressing GFP (B16F10-GFP ) were selected by growth on puromycin-containing medium. Approximately 95% of B16F10 melanoma cells were GFP-positive when analyzed by FACS. To induce senescence, B16F10-GFP cells were treated with 7.5 μM docetaxel (DTX) for 3 days and then allowed to recover in normal growth medium for 4 days. To quantify gene expression of senescence markers and NK cell ligands, docetaxel-treated B16F10-GFP cells (B16F10-GFP-SNC) were homogenized with 1 mL of Trizol (Thermo Fischer) by using a homogenizer. Homogenized cells were transferred to a new Eppendorf tube. Total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using QuantiTect reverse transcription (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in expression levels. The expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct target−Ct18S. Expression of different genes is plotted as fold change in B16F10-GFP-SNC cells compared to untreated B16F10-GFP cells.

As shown in FIG. 244, real-time PCR analysis revealed that B16F10-GFP cells treated in vitro with docetaxel increased genes for senescence markers p21, H2AX, and IL6, and NK cell ligands Rae-1e and ULBP-1. Expression was shown to be upregulated when compared to untreated B16F10-GFP cells.

To determine whether chemotherapy-induced senescent tumor cells are reduced by immunotherapy in vivo, B16F10 parental melanoma cells (0.75×10 ⁶ ) were mixed with B16F10-GFP-SNC cells (0.75×10 ⁶ ). and injected the cell mixture subcutaneously in C57BL/6 mice. Mice were also injected with B16F10 and B16F10-GFP cells as controls. B16F10 parental cells grow to form tumors and B16F10-GFP-SNC cells become part of the tumor microenvironment. When tumors reached approximately 350 mm ³ , mixed tumor-bearing mice were divided into two groups. One group received PBS as a control and the other group received subcutaneous TGFRt15-TGFRs (3 mg/kg) containing TA99 (200 μg). Mice were sacrificed 4 days after immunotherapy treatment. To determine tumor-infiltrating immune cells, tumor tissue was dissociated into single cell suspensions by collagenase digestion. Single cell suspensions were layered onto Ficoll-Paque medium followed by density gradient centrifugation to separate lymphocytes and tumor cells. Cells were centrifuged at 1000 g for 20 minutes at 20° C. with slow acceleration and break off. After centrifugation, Ficoll-Paque clearly separates the two layers. TILs are found at the interface between medium and Ficoll-Paque, while the pellet is composed of tumor cells. TILs were carefully removed from the interface and washed with complete RPMI medium. After washing, RBCs were lysed in ACK buffer for 5 minutes at room temperature. The remaining cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells within the tumor, cells were stained with antibodies specific for cell surface CD3, CD45, CD8, and NK1.1 (BioLegend) for 30 minutes at room temperature. After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). After two washes, cells were resuspended in fixation buffer. After fixation, cells were washed and treated with permeabilization buffer (Invitrogen) for 20 min at 4° C. followed by washing with permeabilization buffer (Invitrogen). Cells were then stained for an intracellular marker for proliferation (Ki67) for 30 minutes at room temperature. After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in FIG. 245, in tumors after TGFRt15-TGFRs+TA99 treatment, the percentage of CD8 ⁺ T cells and natural killer (NK) cells increased after 4 days of treatment compared to controls. These results demonstrate that TGFRt15-TGFRs can stimulate the infiltration of CD8 ⁺ T cells and NK cells in tumors. Both CD8 ⁺ T cells and NK immune cells were also able to proliferate within the tumor as measured by the Ki67 marker.

To determine whether chemotherapy-induced senescent tumor cells are reduced by immunotherapy in vivo, B16F10 parental melanoma cells (0.75×10 ⁶ ) were mixed with B16F10-GFP-SNC cells (0.75×10 ⁶ ). and injected the cell mixture subcutaneously in C57BL/6 mice. Mice were also injected with B16F10 and B16F10-GFP cells as controls. B16F10 parental cells grow to form tumors and B16F10-GFP-SNC cells become part of the tumor microenvironment. When tumors reached approximately 350 mm ³ , mixed tumor-bearing mice were divided into two groups. One group received PBS as a control and the other group received subcutaneous TGFRt15-TGFRs (3 mg/kg) containing TA99 (200 μg). Mice were sacrificed on days 4 and 10 after immunotherapy. To determine tumor-infiltrating immune cells and GFP-positive cells within the tumor, tumor tissue was dissociated into single cell suspensions by collagenase digestion. Flow cytometry analysis of tumor cells (Fig. 246A) showed that immunotherapy-treated mice had lower numbers of GFP-positive cells at days 4 and 10 after treatment compared to the PBS control group. showed that Tumor cells were seeded in 24-well plates and evaluated by fluorescence microscopy (Fig. 246B).

Microscopic images also showed fewer GFP-positive cells in the tumors of immunotherapy-treated mice compared to the control PBS-treated group. Since GFP expression in tumors is associated with chemotherapy-induced B16F10-GFP senescent cells, reduction of GFP expression after immunotherapy treatment indicates successful elimination of senescent tumor cells in tumor-bearing mice. .

Example 113: TGFβ Levels in Kidneys After Induction of Renal Injury by Treatment with Cisplatin and TGFRt15-TGFRs by Tissue ELISA. Collected to assess changes in levels. Eight-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were injected with cisplatin (5 mg/kg, ip) weekly for 3 weeks to induce renal 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 and kidneys were collected and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Samples were homogenized with 0.3 mL of extraction buffer (Abcam) using a homogenizer. The homogenized tissue was transferred to a new Eppendorf tube. Protein levels in homogenized tissues were quantified using the BCA Protein assay kit (Pierce). A mouse TGFβ ELISA (R&D System) was performed on 200 μg of tissue. Concentrations of TGFβ were calculated per milligram of tissue.

As shown in Figure 247, TGFβ levels were decreased in the kidneys of mice treated with TGFRt15-TGFRs compared to the PBS control group. These results indicate that TGFRt15-TGFRs treatment can provide long-lasting activity in reducing TGFβ levels in tissues of chemotherapy-treated mice.

Example 114: Toxicity of Subcutaneous Administration of TGFRt15-TGFRs in Mice To further evaluate the dose-dependent toxicological effects of TGFRt15-TGFRs, female C57BL/6 mice (N=3/group) were injected with PBS or TGFRt15- TGFRs were administered subcutaneously once or twice (every two weeks) at 3, 10, 50 and 200 mg/kg. Animals were monitored for signs of study drug-related toxicity, body weight changes during the study, and hematology and serum chemistry parameters on day 7 after dosing. Mice receiving 200 mg/kg TGFRt15-TGFRs began 4 days after the first injection (study day (SD) 0) and reached a nadir between SDs 6-9, followed by pre-dose by SD11. showed a significant weight loss before returning to the level of . Death was observed in one mouse in the 200 mg/kg group at SD9. No apparent treatment-related effects on body weight or other clinical signs were present in the other dose groups or after the second dose of TGFRt15-TGFRs at 200 mg/kg. After one or two doses of TGFRt15-TGFRs, spleen weight increased in a dose-dependent manner (FIG. 248B). Compared to the PBS group, mice showed a 25-fold increase in WBC counts 7 days after a single 200 mg/kg dose of TGFRt15-TGFRs and remained 5-fold higher 7 days after a second 200 mg/kg dose. There was (Figure 248C, Tables 3 and 4). WBC subset analysis showed a 16-fold increase in absolute lymphocyte counts and a >50-fold increase in neutrophil, monocyte, eosinophil, and basophil counts at SD7 in the 200 mg/kg group. These changes were not observed at lower dose levels of TGFRt15-TGFRs, but were similar to those reported in C57BL/6 mice treated with IL-15/IL-15Rα complex subcutaneously ( Liu et al., Cytokine 107:105-112, 2018). Other hematology and serum chemistry parameters were similar in TGFRt15-TGFRs and PBS-treated animals and generally within the expected range for C57BL/6 mice (Tables 3 and 4). The TGFRt15-TGFRs-mediated effect was maximal 7 days after the first dose and diminished after the second dose, indicating a diminished immune response in mice after repeated doses of IL-15/IL-15Rα. (Elpek et al., PNAS107:21647-21652, 2010; Frutoso et al., J Immunol 201:493-506, 2018). Overall, TGFRt15-TGFRs were well tolerated by C57BL/6 mice at dose levels up to 50 mg/kg.

Table 3. Hematology and Serum Chemistry Parameters of C57BL/6 Mice on Study Day 7 After Single Administration of TGFRt15-TGFRs

Table 4. Hematology and serum chemistry parameters of C57BL/6 mice on study day 21 after two doses of TGFRt15-TGFRs

Example 115: Sequestration of TGF-β by TGFRt15-TGFRs and TGFRt15*-TGFRs in Mice Female C57BL/6 mice were injected subcutaneously with PBS or 3 mg/kg of TGFRt15-TGFRs or TGFRt15*-TGFRs and treated with various doses. Plasma was collected at time points. Plasma levels of TGF-β1 and TGF-β2 were determined using the TGFβ3-Plex assay (Eve Technologies, Calgary, Ala., Canada). TGFRt15-TGFRs and TGFRt15*-TGFRs were found to significantly decrease plasma TGF-β1 and TGF-β2 levels in C57BL/6 mice after 2 days of treatment (FIG. 249), suggesting that TGFβRII of these fusion proteins consistent with domain activity.

Example 116 Effects of TGFRt15-TGFRs and TGFRt15*-TGFRs on Immune Cell Metabolism In Vivo and In Vitro Extracellular flux assays were performed on splenocytes isolated from mice 4 days after administration of 15/IL-15R (IL15SA). Mouse splenocyte extracellular flux assays were performed using the XFp analyzer (Seahorse Bioscience). As expected, TGFRt15-TGFRs and IL-15 dose-level-dependently increased the percentage of glycolytic capacity (ECAR) (FIG. 250A) and mitochondrial respiratory capacity (OCR) (FIG. 250B) of isolated splenocytes. rice field. In vivo TGFRt15*-TGFRs treatment also increased ECAR and OCR of splenocytes. This phenomenon was not observed when splenocytes from untreated C57BL/6 mice were incubated in vitro with TGFRt15*-TGFRs for 4 days. Only TGFRt15-TGFRs (but not TGFRt15*-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 TGFβRII domains of TGFRt15-TGFRs play a role in stimulating immune cell metabolism in vivo.

Example 117: Antitumor Effects of TGFRt15-TGFRs and TGFRt15*-TGFRs Against B16F10 Melanoma in C57BL/6 Mice To evaluate the antitumor effects of TGFRt15-TGFRs and TGFRt15*-TGFRs , a mouse B16F10 tumor model was selected. rice field. It is highly aggressive, poorly immunogenic, lacks immune infiltration, expresses TGF-β that plays a role in its growth, and is resistant to cytokine and checkpoint blocking immunotherapy. B16F10 melanoma cells (5×10 ⁵ cells) (CRL-6475, ATCC) were injected subcutaneously into C57BL/6 mice followed by PBS, TGFRt15-TGFRs (3 or 20 mg/kg) or TGFRt15*-TGFRs (3 or 20 mg/kg). kg) injected subcutaneously on days 1 and 4 after tumor implantation. Tumor volumes were measured every other day and mice with tumors larger than 4000 mm ³ were sacrificed according to IACUC regulations. Survival of mice was also assessed during the study period. When compared through SD15 (ie, before animal death), treatment with 20 mg/kg TGFRt15-TGFRs or TGFRt15*-TGFRs resulted in significantly slower tumor growth than observed in PBS-treated mice (FIG. 252A). Tumor-bearing mice treated with 20 mg/kg TGFRt15-TGFRs also showed prolonged survival when compared to 3 mg/kg TGFRt15-TGFRs and PBS-treated groups (FIG. 252B). These results indicate that TGFRt15-TGFRs and TGFRt15*-TGFRs have antitumor activity against solid B16F10 melanoma tumors, and that the bifunctional TGFRt15-TGFRs conjugate exhibits greater efficacy. Thus, both the TGFβRII and IL-15/IL-15RαSu domains play a role in TGFRt15-TGFRs-mediated activity against B16F10 tumors.

TGFRt15-TGFRs treatment can significantly increase the number of NK and T cells in vivo. To determine whether these immune cells are involved in TGFRt15-TGFRs-mediated anti-tumor effects, we tested CD8 ⁺ T cells and NK1.1 ⁺ cells in tumor-bearing mice prior to TGFRt15-TGFRs treatment with Ab Immunodepletion was performed. Depletion of NK1.1 ⁺ cells (alone or in combination with depletion of CD8 ⁺ T cells) abrogates the anti-tumor effect of TGFRt15-TGFRs during the first two weeks after treatment in B16F10 tumor-bearing mice (FIG. 252C), but either NK1.1 ⁺ cell depletion or CD8 ⁺ T cell depletion reduced the survival benefit seen with TGFRt15-TGFRs (FIG. 252D). Consistent with these findings, TGFRt15-TGFRs treatment also promoted increased NK cell and CD8 ⁺ T cell infiltration into B16F10 tumors (FIG. 252E). These results support the conclusion that both CD8 ⁺ T cells and NK cells play a major role in TGFRt15-TGFRs-mediated activity against melanoma tumor cells in C57BL/6 mice.

Example 118: TGFRt15-TGFRs improved glucose control in db/db mice, 5-week-old male BKS. Cg-Dock7m+/+Leprdb/J (db/db) mice (Jackson Lab) were fed standard chow and maintained under standard conditions. Mice (n=5/group) received subcutaneous injections of either PBS (control group) or TGFRt15-TGFRs (3 mg/kg) (treatment group) at 6 and 12 weeks from the start of the study. Fasting blood glucose and insulin were checked 3 weeks after the first administration. After TGFRt15-TFGRs treatment, fasting blood glucose was significantly reduced compared to controls (FIG. 253A), but blood insulin levels were unchanged (FIG. 253B).

Example 119: TGFRt15-TGFRs significantly downregulated senescence index and SASP index in 5-week-old male BKS. Cg-Dock7m+/+Leprdb/J (db/db) mice were fed a standard chow diet and had access to drinking water ad libitum. At 6 weeks of age, mice were randomly assigned to control and treatment groups (n=5/group). The treatment group received TGFRt15-TGFRs by subcutaneous injection at 3 mg/kg at weeks 6 and 12 from study initiation, while the control group received vehicle only (PBS). At the end of the study (4 weeks after the second dose) mice were euthanized and pancreases were harvested. Half of the pancreas was homogenized with TRIzol reagent (Invitrogen) and total tissue RNA was purified with the RNeasy mini kit (Qiagen). Synthesis of cDNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen) and quantitative PCR was performed with SsoAdvanced™ Universal SYBR® Green Supermix (BioRad) according to the comparative threshold cycle method according to the manufacturer's protocol. and performed using the QuantiStudio 3 real-time PCR system (Applied Biosystems). Amplification reactions were performed in duplicate and fluorescence curves were analyzed with the software provided with the QuantiStudio 3 real-time PCR system. Housekeeping gene 18s ribosomal RNA was used as an endogenous control. Expression of each target mRNA relative to 18s rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct _target −Ct _18S . As shown in FIG. 254A, TGFRt15-TGFRs treatment of db/db mice reduced senescence gene indices of p16, p21, Igfr1, and Bamb1, and SASP gene indices of IL-1α, IL-1, and IL-1 when compared to the control group. 6, MCP-1, and TNFα resulted in decreased pancreatic gene expression. In general, pancreatic expression of genes for SASP index and senescence index was significantly reduced after TGFRt15-TGFRs treatment compared to controls, whereas pancreatic gene expression for beta-cell index was significant in TGFRt15-TGFRs and PBS-treated db/db. (Figures 254B, 254C, 254D). The data suggest that TGFRt15-TGFRs possess potent senolytic properties and senescent cell inhibitory activity to reduce pancreatic senescent cells and SASP factors in db/db mice.

Example 120: TGFRt15-TGFRs reduced pancreatic beta-cell senescent cells in 5-week-old male BKS. Cg-Dock7m+/+Leprdb/J (db/db) mice were fed a standard chow diet (Irradiated 2018 Teklad global 18% protein rodent diet, Envigo) and received drinking water ad libitum. At 6 weeks of age, mice were randomly assigned to control and treatment groups (n=5/group). The treatment group received TGFRt15-TGFRs by subcutaneous injection at 3 mg/kg at weeks 6 and 12 from study initiation, while the control group received vehicle only (PBS). At the end of the study (4 weeks after the second dose), mice were euthanized and pancreata were removed en bloc and immersion fixed in 4% formaldehyde (4% formaldehyde in 0.1 M phosphate buffer, PBS pH 7.4). , and stored at 4°C until further processing. Dissected pancreases were paraffinized, embedded, sectioned, and three 10 mm sections (150 mm apart) were cut from each block, totaling to represent a systematic, uniform, random sample of whole pancreas from each animal.

Multispectral imaging was performed using an Akoya Vectra Polaris instrument. This instrument allows phenotypic analysis, quantification, and spatial relationship analysis of tissue invasion in formalin-fixed, paraffin-embedded biopsy sections. To quantify levels of p21 in insulin+ islet regions of the pancreas, formalin-fixed, paraffin-embedded tissue sections were serially treated with specific primary antibodies according to standard protocols provided by Akoya and routinely performed by HIMSR. specifically dyed. Briefly, sections were deparaffinized, heat treated with antigen retrieval buffer, blocked, rabbit primary antibodies against insulin (#4590, Cell Signaling Technology) and p21 (EPR362, Abcam) followed by horseradish peroxidase (HRP). Incubated with a conjugated secondary antibody polymer (anti-rabbit), HRP-reactive OPAL fluorescent reagents (OPAL-520 for insulin, OPAL-570 for p21, Akoya) were labeled for each HRP molecule using TSA chemistry. It pigments the tissue in the immediate surroundings. Sections were stripped and heat treated with antigen retrieval buffer (citrate buffer for insulin, EDTA buffer for p21) between each staining to prevent further deposition of fluorescent dye in subsequent staining steps. gone. Whole-section scans were collected on an Akoya Vectra Polaris instrument using a 20× objective with a resolution of 0.5 microns. Three color images were analyzed with inForm software (Akoya) to separate adjacent fluorochromes, subtract autofluorescence, segment insulin ⁺ regions of tissue, compare cell frequency and location, and analyze cell cytoplasm and We segmented the nuclear region and phenotyped the infiltrating cells by cell marker expression.

As shown in Figures 255A-255D, p21-positive senescent cells (OPAL-570) accumulated more in insulin-positive islet beta cells (OPAL-520) in the control pancreas (Figure 255A), indicating that TGFRt15- These senescent cells were reduced in the pancreas of the 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). p21-positive senescent beta cells (insulin positive) were reduced in the TGFRt15-TGFRs treated group compared to the control group, but the difference was not statistically significant (FIG. 255D). Overall, the data suggest that TGFR15-TGFRs have senolytic 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 reduced pancreatic beta-cell senescent cells by increasing NK, NKT, and CD8+ T cells in 5-week-old male BKS. Cg-Dock7m+/+Leprdb/J (db/db) mice were fed a standard chow diet (Irradiated 2018 Teklad global 18% protein rodent diet, Envigo) and received drinking water ad libitum. At 6 weeks of age, mice were randomly assigned to control and treatment groups (n=5/group). The treatment group received TGFRt15-TGFRs by subcutaneous injection at 3 mg/kg at weeks 6 and 12 from study initiation, while the control group received vehicle only (PBS).

Four days after the first dose treatment, blood was collected and whole blood cells (50 mL) were treated with ACK (ammonium-chloride-potassium) lysis buffer to lyse red blood cells. Lymphocytes were then stained with PE-Cy7-anti-CD3, BV605-anti-CD45, PerCP-Cy5.5-anti-CD8a, BV510-anti-CD4, and APC-anti-NKp46 antibodies (all from BioLegend). to assess T cell, NKT cell, and NK cell populations. As shown in Figures 256A-256C, after treatment with TGFRt15-TGFRs, the percentages of CD8+ T cells, CD3 ⁺ NKP46 ⁺ NKT cells, and CD3 ^- NKP46 ⁺ NK cells in the blood of db/db mice were compared with those of PBS-treated mice. increased compared to

Example 122 Phenotyping of Immune Cell Subsets in Peripheral Blood of Cynomolgus Monkeys Following Administration of TGFRt15-TGFRs /kg of PBS (vehicle) or TGFRt15-TGFRs subcutaneously. Blood was collected the day before treatment (Day 1) and 5, 22, and 29 days after treatment. PBMC were prepared and stained with a panel of fluorescently labeled antibodies to assess the phenotype of B cells, NK cells, NK-T cells, Treg cells, CD4 ⁺ and CD8 ⁺ T cells by flow cytometry. Figure 257 shows that administration of TGFRt15-TGFRs significantly increased the percentage of Ki67 ⁺ NK cells, NK-T cells, Treg cells and CD4 ⁺ and CD8 ⁺ T cells 5 days after treatment. These findings indicate that TGFRt15-TGFRs treatment induced proliferation of these lymphocyte subsets in non-human primates. No treatment effect on Ki67 expression in B cells was observed.

Example 123: IL-15 Immunostimulatory and TGF-β Antagonist Activity of TGFRt15-TGFRs In 6-week-old (young) and 72-week-old (old) C57BL/6 mice, PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15*-TGFRs (3 mg/kg) were injected subcutaneously as a single dose. Mice were sacrificed 4 days after treatment and spleens were collected. The spleen was crushed with the flat back end of a sterile piston/plunger of a 3cc syringe to release the splenocytes. Splenocytes were passed through a 70 μM cell strainer and homogenized into a single cell suspension. RBCs were lysed with ACK lysis buffer and splenocytes were washed and counted. To measure splenocyte glycolytic activity, cells were washed, resuspended in Seahorse medium, and resuspended at 4×10 ⁶ cells/mL. Cells were seeded at 50 μL/well in Cell-Tak coated Seahorse Bioanalyzer XFe96 culture plates in Seahorse XF RPMI medium pH 7.4 supplemented with 2 mM L-glutamine for glycolytic stress studies. Cells were allowed to attach to the plates for 30 minutes at 37°C. Additionally, 130 μL of assay medium was added to each well of the plate (also background wells). Plates were incubated for 1 hour at 37°C, non- _CO2 incubator. For the glycolytic stress test, the calibration plate contained a 10x solution of glucose/oligomycin/2DG prepared in Seahorse assay medium and 20 μL of glucose/oligomycin/2DG was calibrated overnight. was added to each port of the extracellular flux plate. The glycolytic stress test is based on extracellular acidification rate (ECAR) and measures three key parameters of glycolytic function including glycolysis, glycolytic capacity, and glycolytic reserve. A complete ECAR assay was performed in four phases: non-glycolytic acidification (no drug), glycolytic (10 mM glucose), maximal glycolytic induction/glycolytic capacity (2 μM oligomycin), and glycolytic reserve (100 mM 2-DG). consists of stages. At the end of the experiment, data were exported as Graph Pad Prism files. The XF Glycolytic Stress Test Report Generator automatically calculated the parameters of the XF Cellular Glycolytic Stress Test from the Wave data. Data were analyzed using Wave software (Agilent).

As shown in FIG. 258, splenocytes isolated from aged mice 4 days after TGFRt15-TGFRs treatment showed higher basal glycolysis, glycolytic showed enhanced potency and glycolytic reserve. Glycolytic function of splenocytes in old control mice was lower than in young control mice. Treatment of young and aged mice with TGFRt15*-TGFRs was able to increase the glycolytic function of splenocytes. However, TGFRt15-TGFRs treatment of aged mice reduced the basal glycolytic rate, glycolytic capacity, and glycolytic reserve of splenocytes to levels similar to those observed in splenocytes from TGFRt15-TGFRs-treated young mice. could be increased to These findings suggest that IL-15 immunostimulation and TGF-β antagonistic activity of TGFRt15-TGFRs effectively stimulate and rejuvenate the decreased metabolic activity of immune cells in aged mice.

Six-week-old (young) and 72-week-old (old) C57BL/6 mice were injected subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg) or TGFRt15*-TGFRs (3 mg/kg) in a single dose. Mice were sacrificed 4 days after treatment and spleens were collected. The spleen was crushed with the flat back end of a sterile piston/plunger of a 3cc syringe to release the splenocytes. Splenocytes were passed through a 70 μM cell strainer and homogenized into a single cell suspension. RBCs were lysed with ACK lysis buffer and splenocytes were washed and counted. To measure splenocyte mitochondrial respiration, cells were washed, resuspended in Seahorse medium, and resuspended at 4×10 ⁶ cells/mL. Cells were seeded at 50 μL/well in Cell-Tak coated Seahorse Bioanalyzer XFe96 culture plates in Seahorse XF RPMI medium pH 7.4 supplemented with 2 mM L-glutamine for glycolytic stress studies. For mitochondrial stress tests, cells were seeded in Seahorse XF RPMI medium, pH 7.4, supplemented with 10 mM glucose and 2 mM L-glutamine. Cells were allowed to attach to the plates for 30 minutes at 37°C. Additionally, 130 μL of assay medium was added to each well of the plate (also background wells). Plates were incubated for 1 hour at 37°C in a non-CO2 incubator. For the mitochondrial stress test, the calibration plate contained a 10x oligomycin/FCCP/rotenone solution prepared in Seahorse assay medium and 20 μL of oligomycin, FCCP, and rotenone were calibrated overnight. was added to each port of the extracellular flux plate. Oxygen consumption rate (OCR) was measured using an XFe96 Extracellular Flux Analyzer. A complete OCR analysis consisted of four stages: basal respiration (no drug), ATP-related respiration/proton leak (1.5 μM oligomycin), maximal respiration (2 μM FCCP), and pre-respiration (0.5 μM rotenone). . At the end of the experiment, data were exported as Graph Pad Prism files. The XF Mitochondrial Stress Test Report Generator automatically calculates the parameters of the XF Mitochondrial Stress Test from Wave data exported to Excel. Data were analyzed by using Wave software (Agilent).

As shown in FIG. 259, splenocytes isolated from aged mice on day 4 after TGFRt15-TGFRs therapy showed increased basal respiration, ATP-related respiration, Showed maximal respiration and increased reserve capacity. Treatment of young and aged mice with TGFRt15*-TGFRs was able to increase splenocyte mitochondrial respiration. However, TGFRt15-TGFRs treatment of aged mice increased the rate of basal glycolysis, ATP-related respiration, maximal respiration and reserve capacity, at levels comparable to or greater than those observed in splenocytes from TGFRt15-TGFRs-treated young mice. could be increased to the level of These findings suggest that IL-15 immunostimulation and TGF-β antagonistic activity of TGFRt15-TGFRs effectively stimulate and rejuvenate the decreased metabolic activity of immune cells in aged mice.

Example 124: TGFRt15-IL-15 activity of TGFRs plays a role in the expansion of CD8+ T cells and NK cells 6-week-old (young) and 72-week-old (old) C57BL/6 mice were purchased from Jackson Laboratory . Mice were housed in a temperature and light controlled environment. Mice (n=6/group) were treated subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg) and TGFRt15*-TGFRs (3 mg/kg). To assess changes in different subsets of immune cells, mouse blood was collected from the submandibular vein 4 days after treatment with tubes containing EDTA. Whole blood RBCs were lysed in ACK buffer for 5 minutes at room temperature. The remaining cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess different types of immune cells in the fluid, cells were stained with antibodies specific for cell surface CD3, CD4, CD45, CD8, and NK1.1 (BioLegend) for 30 min at room temperature (RT). After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figure 260, the results showed that treatment of aged mice with TGFRt15-TGFRs induced an increase in the percentage of CD3 ⁺ CD45 ⁺ , CD3 ⁺ CD8 ⁺ , and CD3 ⁻ NK1.1 ⁺ immune cells in the blood. However, treatment of aged mice with TGFRt15*-TGFRs did not affect the percentages of these blood cell populations. These results suggest that IL-15 activity of TGFRt15-TGFRs plays a role in increasing CD8 ⁺ T cells and NK cells in the blood of aged mice. The percentage of circulating T cells and NK cells in old control mice was lower than in young control mice. However, treatment of aged mice with TGFRt15-TGFRs decreased the percentage of CD3 ⁺ CD45 ⁺ , CD3 ⁺ CD8 ⁺ , and CD3 ⁻ NK1.1 ⁺ immune cells in the blood to that of young mice treated with TGFRt15-TGFRs. It was increased to levels similar to those observed.

6-week-old (young) and 72-week-old (old) C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice (n=6/group) were treated subcutaneously with PBS, TGFRt15-TGFRs (3 mg/kg) and TGFRt15*-TGFRs (3 mg/kg). Four days after treatment, mice were euthanized and spleens were harvested and processed into single cell suspensions. Single cell suspensions were prepared to evaluate different subsets of immune cells. RBCs were lysed in ACK buffer for 5 minutes at room temperature. The remaining cells were washed in FACS buffer containing 1×PBS (Hyclone), 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma). To assess the different types of immune cells in the spleen, cells were stained with antibodies specific for cell surface CD3, CD45, CD8, and NK1.1 (BioLegend) for 30 minutes at room temperature. After surface staining, cells were washed in FACS buffer (1×PBS (Hyclone) containing 0.5% BSA (EMD Millipore) and 0.001% sodium azide (Sigma)) (1500 RPM, 5 minutes at room temperature). minutes). After washing twice, cells were analyzed by flow cytometry (Celesta-BD Bioscience).

As shown in Figure 261, the results showed that treatment of aged mice with TGFRt15-TGFRs induced an increase in the percentage of CD3 ⁺ CD45 ⁺ , CD3 ⁺ CD8 ⁺ , and CD3 ⁻ NK1.1 ⁺ immune cells in the spleen. However, treatment of aged mice with TGFRt15*-TGFRs did not affect the percentage of these splenocyte populations. These results suggest that IL-15 activity of TGFRt15-TGFRs plays a role in increasing CD8 ⁺ T cells and NK cells in the blood of aged mice. The percentage of splenic T cells and NK cells in old control mice was lower than in young control mice. However, treatment of old mice with TGFRt15-TGFRs decreased the percentage of CD3 ⁺ CD45 ⁺ , CD3 ⁺ CD8 ⁺ , and CD3 ⁻ NK1.1 ⁺ immune cells in the spleen to that of young mice treated with TGFRt15-TGFRs. It was increased to levels similar to those observed.

Example 125: TGFRt15-TGFRs Associated Decrease in Spontaneous Senescent Cells in the Liver 72-week-old (aged) C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice (n=8/group) were treated subcutaneously with either 1 dose or 2 doses (days 0 and 60) of PBS or TGFRt15-TGFRs (3 mg/kg). Seventy-one days after treatment, mice were euthanized and livers were collected and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Tissue samples were homogenized with 1 mL of Trizol (Thermo Fischer) using a homogenizer. Homogenized tissue was transferred to a new Eppendorf tube and total RNA was extracted using the RNeasy mini kit (Qiagen #74106) according to the manufacturer's instructions. 1 μg of total RNA was used for cDNA synthesis using QuantiTect reverse transcription (Qiagen). Real-time PCR was performed on a CFX96 detection system (Bio-Rad) using FAM-labeled pre-designed primers purchased 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 variations in gene expression levels. Expression of each target mRNA relative to 18S rRNA was calculated as 2 ^{− Δ(ΔCt)} based on Ct, where ΔCt=Ct _target −Ct _18S . Untreated 6-week-old mice were used as controls to compare gene expression levels with aged mice. Results showed that gene expression of IL-1α, IL-1β, IL-6, p21, and PAI-1 in the liver increased with age in mice, as expected with an age-dependent increase in senescence-associated transcripts. shown to increase. Treatment of 72-week-old mice with 1 or 2 doses of TGFRt15-TGFRs increased the hepatic senescence markers IL-1α, IL-1β, IL-6, p21, and PAI-1 when compared to the PBS control group. gene expression was significantly reduced (Fig. 262). These findings suggest a TGFRt15-TGFRs-associated reduction of spontaneous senescent cells in the liver of aged mice.

Example 126: TGFRt15-TGFRs Treatment Can Reduce Hepatic Tissue Inflammation 72-week-old (senile) C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice (n=10/group) were treated subcutaneously with either one or two doses of PBS or TGFRt15-TGFRs (3 mg/kg). 120 days after treatment, mice were euthanized and mouse livers were prepared for evaluation by histochemistry. Liver tissue specimens were fixed with 10% formaldehyde and sections were stained with hematoxylin-eosin after a paraffin blocking procedure. The degree of liver injury was assessed histologically in a blinded fashion. Histological sections of whole liver were scored for inflammation using a scale of 0 to 4 (0, absent and normal-appearing, 1, mild, 2, moderate, 3, strong, and 4, strong). As shown in Figure 263, two doses of TGFRt15-TGFRs reduce liver inflammation scores in livers of aged mice compared to single doses of TGFRt15-TGFRs or PBS control group. These results suggest that TGFRt15-TGFRs treatment can reduce inflammation in liver tissue of aged mice.

Example 127: TGFRt15-TGFRs Treatment Can Reduce IL1-α, IL-6, IL-8, PAI-1 and Fibronectin Protein Levels 72-week-old (senile) C57BL/6 mice from Jackson Laboratory Purchased. Mice were housed in a temperature and light controlled environment. Mice (n=10/group) were treated with either one or two doses (days 0 and 60) of PBS or TGFRt15-TGFRs (3 mg/kg). 120 days after treatment, mice were euthanized and livers were collected and stored in liquid nitrogen in 1.7 mL Eppendorf tubes. Tissue samples were homogenized with 0.3 mL of extraction buffer (Abcam) using a homogenizer. The homogenized tissue was transferred to a new Eppendorf tube. Protein levels in homogenized tissues were quantified using the BCA Protein assay kit (Pierce). ELISAs (R&D System) for detection of IL-1α, IL-1β, IL-6, IL-8, TGF-β, PAI-1, collagen and fibronectin were performed using 25 μg tissue homogenization. As shown in FIG. 264, protein levels of IL-1α, IL-6, IL-8, PAI-1, and fibronectin increased compared to single doses of PBS control or TGFRt15-TGFRs treated groups compared to TGFRt15- It was reduced in the liver of mice treated with two doses of TGFRs. These results indicate that two doses of TGFRt15-TGFRs treatment can reduce hepatic IL-1α, IL-6, IL-8, PAI-1, and fibronectin protein levels in aged mice. IL-1β, TGF-β, and collagen protein levels were also lower in livers of mice treated with two doses of TGFRt15-TGFRs compared to PBS controls. However, these changes did not reach statistical significance.

Example 128: TGFRt15-TGFRs Reduce Senescent Cells 72-week-old (aged) C57BL/6 aged mice purchased from Jackson Laboratory were housed in a temperature and light controlled environment. Mice (n=5/group) were treated subcutaneously with either PBS or TGFRt15-TGFRs (3 mg/kg). Mice were euthanized 4 days after treatment and livers were collected, homogenized with FBS containing 2% PBS and filtered through a 70 micron filter to obtain a single cell suspension. Cells were spun down and resuspended in 5 mL RPMI containing 0.5 mg/mL collagenase IV and 0.02 mg/mL DNAse in a 14 mL round bottom tube. Cells were then shaken on an orbital shaker at 37° C. for 1 hour and washed twice with RPMI. Cells were cultured in 2 mL complete medium (supplemented with RPMI 1640 (Gibco), 2 mM L-glutamine (Thermo Life Technologies), penicillin (Thermo Life Technologies), streptomycin (Thermo Life Technologies), and 10% FBS (Hyclone)). inside Resuspended at 2×10 ⁶ /mL in 24-well flat bottom plates and incubated at 37° C., 5% CO ₂ for 48 hours. Cells were harvested and washed once with warm complete medium at 1000 rpm for 10 minutes at room temperature. Cell pellets were resuspended in 500 μL of fresh medium containing 1.5 μL of senescence dye (Abcam) per tube. Cells were further incubated at 37° C., 5% CO ₂ for 1-2 hours and washed twice with 500 μL of wash buffer. Pellets were resuspended in 500 μL of wash buffer and immediately analyzed by flow cytometry (Celesta-BD Bioscience). As shown in Figure 265, the percentage of senescence marker β-gal ⁺ cells decreased after 4 days of in vivo treatment with TGFRt15-TGFR. These results demonstrate that TGFRt15-TGFRs can reduce hepatic senescent cells (based on the β-gal marker) in aged mice.

Example 129 Effect of TGFRt15-TGFRs on Survival of Aged Mice 72-week-old C57BL/6 mice were purchased from Jackson Laboratory. Mice were housed in a temperature and light controlled environment. Mice were treated subcutaneously with either PBS or a single dose of TGFRt15-TGFRs (3 mg/kg) (n=20/group). Mice were monitored daily for survival up to 120 weeks after treatment. The survival probabilities of treatment groups based on the Mantel-Cox log-rank test are shown in FIG. Higher mortality was seen in control mice compared to TGFRt15-TGFRs, which was represented by decreased mouse survival. By 120 weeks post-treatment, PBS control mice had a mortality rate of 70% compared to 45% mortality in mice treated with TGFRt15-TGFRs.

Example 130: Effect of TGFRt15-TGFRs in reducing SASP factors in the liver of B16F10 tumor-bearing mice after chemotherapy The effect of TGFRt15-TGFR treatment in reducing protein levels of SASP factors in B16F10 tumor-bearing mice after chemotherapy was further investigated. evaluated. Mice were implanted on day 0 with B16F10 tumor cells (1×10 ⁷ cells/mouse). Mice were treated subcutaneously on days 1, 4, and 7 with 10 mg/kg docetaxel. On day 8, mice were dosed subcutaneously with PBS or TGFRt15-TGFRs (3 mg/kg). Mice were euthanized 17 days after tumor inoculation and livers were harvested and homogenized. Protein levels of SASP factors in liver homogenates were determined by ELISA. As shown in Figure 267, in vivo treatment with TGFRt15-TGFRs resulted in a significant reduction in levels of hepatic IL-1α, IL-6, TNFα and IL-8 SASP factors in B16F10 tumor-bearing mice after chemotherapy. .

Example 131: Role of Immune Cell Subsets in TGFRt15-TGFRs-Mediated Senescent Tumor Cell Clearance in B16F10 Melanoma Mouse Model Induced senescent B16F10-GFP tumor cells were mixed with parental B16F10 cells and implanted subcutaneously into mice after treatment with anti-NK1.1 or anti-CD8a antibodies. When tumors reached approximately 350 mm ³ , mice were randomized to receive subcutaneous treatment with PBS or TGFRt15-TGFRs (3 mg/kg) plus TA99 (200 μg). Mice were sacrificed 4 days after treatment and tumors were collected and analyzed. Levels of GFP-positive B16F10-GFP TIS cells and NK and CD8 ⁺ T cells in tumors were assessed by flow cytometry. As shown in FIG. 268A, TGFRt15-TGFRs-treated mixed tumors were either non-immunodepleted or depleted of CD8 ⁺ T immune cells and contained significantly fewer GFP-expressing senescent tumor cells than control-treated mice. board. It was also observed that the tumors of CD8 ⁺ depleted mice were significantly infiltrated by NK cells and the tumors of NK depleted mice were significantly infiltrated by CD8 ⁺ T cells (Fig. 268B). These results suggest that both NK cells and CD8 ⁺ T cells play a role in regulating tumor growth, with NK cells primarily mediating the activity of TGFRt15-TGFRs to deplete TIS tumor cells. there is

Example 132: To further evaluate sequential treatment with anti-PD-L1 antibody TGFRt15-TGFRs-immune checkpoint inhibitor in combination with TGFRt15-TGFRs + TA99 and chemotherapy in B16F10 melanoma mouse model (described in Example 109) , B16F10 tumor-bearing mice were first treated with doxetaxel (DTX) and then either TGFRt15-TGFRs+TA99 followed by anti-PD-L1 antibody or anti-PD-L1 antibody followed by TGFRt15-TGFRs+TA99 (FIG. 269A). processed with Tumor growth curves and endpoint tumor volumes at day 18 showed that both combination strategies (TGFRt15-TGFRs+TA99 followed by anti-PD-L1 and vice versa) were better than individual immunotherapies (TGFRt15-TGFRs+TA99 or anti-PD-L1 alone). either) or DTX alone (FIG. 269B) showed significant tumor volume reduction. Interestingly, TGFRt15-TGFRs+TA99-treated tumors showed significantly lower tumor volume 13 days before the start of combination treatment compared to anti-PD-L1-treated tumors, suggesting that TGFRt15-TGFRs in the early control of tumor growth. The effect of TGFRs+TA99 was shown. Endpoint analysis also showed that tumors treated with the combination of TGFRt15-TGFRs + TA99 and anti-PD-L1 antibody significantly increased levels of tumor-infiltrating CD8+ T cells and NK cells compared to the single treatment group. rice field. Combination treatment increased expression of the co-stimulatory receptor CD28 on CD8 ⁺ TILs compared to single treatment, and checkpoint blockade was further activated by IL-15 activity of TGFRt15-TGFRs within the tumor microenvironment. suggesting that the dysfunctional CD8 ⁺ TILs in the cells can be rescued (Fig. 269C). This was accompanied by an enhanced activation phenotype (IFNγ secretion) of splenic CD8 ⁺ T cells from the combined treatment group after stimulation with PMA/ionomycin (FIG. 269D). Combination treatment also showed increased NKG2D expression on total CD8 ⁺ T cells and CD44 ^-high CD8 ⁺ T cells in tumors compared to individual immunotherapy treatments (FIG. 269E). Collectively, these data indicate that combination therapy of TGFRt15-TGFRs plus TA99 and anti-PD-L1 antibody resulted in activation and infiltration of CD8 ⁺ T cells that may contribute to effective tumor control.

Example 133: Anti-tumor effect of TGFRt15-TGFRs in combination with chemotherapy against SW1990 human pancreatic tumors in C57BL/6SCID mice To further evaluate the anti-tumor activity of TGFRt15-TGFRs in combination with chemotherapy, SW1990 human pancreatic cancer Cells (2×10 ⁶ cells/mouse) were injected subcutaneously (sc) into C57BL/6 scid mice. Nine days after tumor cell implantation, gemcitabine (40 mg/kg, ip) and nab-paclitaxel (Abraxane) (5 mg/kg, ip) chemotherapy was initiated, followed 2 days later by TGFRt15-TGFRs (3 mg/kg, ip). ) was started. This was considered one treatment cycle and was repeated for 3 more cycles (1 cycle/week) (Figure 270A). Tumor-bearing control groups received PBS, chemotherapy, or TGFRt15-TGFRs treatment alone. Tumor volumes were measured during and after study treatment and animal survival was assessed based on tumor volume <4000 mm ³ . Results showed that animals receiving the combination of TGFRt15-TGFRs and chemotherapy had significantly slower SW1990 tumor growth compared to the PBS group (FIGS. 270B-270C). TGFRt15-TGFRs+chemotherapy also prolonged survival of SW1990 tumor-bearing mice (FIG. 270D). These results confirm that TGFRt15-TGFRs enhanced the efficacy of standard-of-care chemotherapy against human pancreatic tumors in mouse xenograft tumor models.

OTHER EMBODIMENTS While the invention has been described in conjunction with the Detailed Description, the foregoing description is intended to be illustrative, the scope of the invention being defined by the appended claims. It should be understood that it is not intended to limit the Other aspects, advantages, and modifications are within the scope of the following claims.

Exemplary Embodiments Embodiment A1. A single-chain chimeric polypeptide,
(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 any one of embodiments A1-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-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-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 second target binding domain.

Embodiment A8. The single-chain 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-A9, wherein the first target binding domain and the second target binding domain specifically bind 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-A9, wherein the first target binding domain and the second target binding domain specifically bind different antigens.

Embodiment A14. The single-chain chimeric polypeptide of any one of embodiments A1-A13, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

Embodiment A15. The single-chain chimeric polypeptide of embodiment A14, wherein the first target binding domain and the second target binding domain are each antigen binding domains.

Embodiment A16. The single-chain chimeric polypeptide of embodiment A13, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment A17. one or both of the first target binding domain and the second target binding domain are 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC , MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122 , CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL -1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-D receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 The single-chain chimeric polypeptide of any one of embodiments A1-A16, which binds a target selected from the group consisting of binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

Embodiment A18. The single-chain chimeric polypeptide of any one of embodiments A1-A16, wherein one or both of the first and second target binding domains is a soluble interleukin or cytokine protein.

Embodiment A19. soluble interleukin, cytokine, or ligand protein is 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 proteins.

Embodiment A20. The single-chain chimeric polypeptide of any one of embodiments A1-A16, wherein one or both of the first and second target binding domains is a soluble interleukin or cytokine receptor.

Embodiment A21. Soluble interleukin or cytokine 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 A22. The single-chain chimeric polypeptide of any one of embodiments A1-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 that is 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 that is 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 that is at least 95% identical to SEQ ID NO:93.

Embodiment A26. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
does not contain one or more of arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein; The method of any one of embodiments A22-A25.

Embodiment A27. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. A method as described in A26.

Embodiment A28. The single-chain chimeric polypeptide of any one of embodiments A1-A27, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment A29. The single-chain chimeric polypeptide of any one of embodiments A1-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-A29, wherein the single chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment A31. The single chain chimeric polypeptide according to any one of embodiments A1-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 chimera of embodiment A32, wherein one or more additional target binding domains are directly adjacent to the first target binding domain, second target binding domain, or soluble tissue factor domain Polypeptide.

Embodiment A34. 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 , a single-chain chimeric polypeptide according to embodiment A33.

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. According to embodiment A35, wherein one of the one or more additional target binding domains is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Single chain chimeric polypeptide.

Embodiment A37. 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 , a single-chain chimeric polypeptide according to embodiment A35.

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. According to embodiment A38, wherein one of the one or more additional antigen binding domains at the N-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Single chain chimeric polypeptide.

Embodiment A40. a single-chain chimeric polypeptide 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; The single-chain chimeric polypeptide of embodiment A38, further comprising a linker sequence.

Embodiment A41. According to embodiment A38, wherein one of the one or more additional antigen binding domains at the C-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Single chain chimeric polypeptide.

Embodiment A42. a single chain chimeric polypeptide 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; The single-chain chimeric polypeptide of embodiment A38, further comprising a linker sequence.

Embodiment A43. Any one of embodiments A31-A42, 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 A single-chain chimeric polypeptide according to .

Embodiment A44. The single chain chimera 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 the same epitope. Polypeptide.

Embodiment A45. The single-chain chimeric polypeptide of embodiment A44, wherein two or more of the first target binding domain, second target binding domain, and 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 the one or more additional target binding domains each specifically bind 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 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 of any one of embodiments A31-A42, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind different antigens. chimeric polypeptide.

Embodiment A50. One according to any one of embodiments A31-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. Main chain chimeric polypeptides.

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 a scFv or single domain antibody.

Embodiment A53. One or both of the first target binding domain and the second target binding domain, and one or more of the target binding domains are 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-D receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, Any one of embodiments A31-A52 that specifically binds to a target selected from the group consisting of MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor A single-chain chimeric polypeptide according to 1.

Embodiment A54. Any one of embodiments A31-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 A single-chain chimeric polypeptide according to 1.

Embodiment A55. soluble interleukin, cytokine, or ligand protein is 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 proteins.

Embodiment A56. Any of embodiments A31-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 1. A single-chain chimeric polypeptide according to one.

Embodiment A57. 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, The single-chain chimeric polypeptide of embodiment A56, which is soluble CD3, or soluble CD28.

Embodiment A58. The single chain chimeric polypeptide of any one of embodiments A1-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-A58, wherein the single chain chimeric polypeptide further comprises a peptide tag positioned at the N-terminus or C-terminus of the single chain chimeric polypeptide.

Embodiment A60. A composition comprising any of the single-chain chimeric polypeptides of embodiments A1-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 of the single-chain chimeric polypeptides of any one of embodiments A1-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, comprising:
culturing the cells of embodiment A66 in a culture medium under conditions sufficient to result in the production of the single-chain chimeric polypeptide;
recovering the single chimeric polypeptide from the cells and/or the culture medium.

Embodiment A68. A single-chain chimeric polypeptide produced by the method of embodiment A67.

Embodiment A69. The single-chain chimeric polypeptide of embodiment A26, wherein the soluble human tissue factor domain comprises a sequence that is 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 that is 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 that is at least 95% identical to SEQ ID NO:97.

Embodiment A72. The single-chain chimeric polypeptide of embodiment A71, wherein the soluble human tissue factor domain comprises a sequence that is 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 that is 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 that is 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 that is at least 95% identical to SEQ ID NO:98.

Embodiment A76. The single-chain chimeric polypeptide of embodiment A75, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:98.

Embodiment B1. A single-chain chimeric polypeptide,
(i) a first target binding domain;
(ii) a soluble tissue factor domain, and (iii) a second target binding domain,
The first target binding domain and the second target binding domain each specifically bind to an IL-2 receptor, or the first target binding domain and the second target binding domain each bind an IL-15 receptor binds specifically to the body,
Single chain chimeric polypeptide.

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 chain chimeric polypeptide of any one of embodiments B1-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-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-chain 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-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 second target binding domain.

Embodiment B8. The single-chain 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-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 soluble human IL-2 protein.

Embodiment B13. The single-chain chimeric polypeptide of embodiment B12, wherein the soluble 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 protein.

Embodiment B15. The single-chain chimeric polypeptide of embodiment B14, wherein the soluble IL-15 protein is 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-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 that is 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 that is 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 that is at least 95% identical to SEQ ID NO:93.

Embodiment B21. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
does not contain one or more of arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein; The method of any one of embodiments B17-B20.

Embodiment B22. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. A method as described in B21.

Embodiment B23. The single-chain chimeric polypeptide of any one of embodiments B1-B22, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment B24. The single-chain chimeric polypeptide of any one of embodiments B1-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-B24, wherein the single chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment B26. The single chain chimeric polypeptide according to any one of embodiments B1-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 chimera of embodiment B27, wherein one or more additional target binding domains are directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Polypeptide.

Embodiment B29. 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 , a single-chain chimeric polypeptide according to embodiment B28.

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. According to embodiment B30, wherein one of the one or more additional target binding domains is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Single chain chimeric polypeptide.

Embodiment B32. 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 , a single-chain chimeric polypeptide according to embodiment B30.

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-terminus and C-terminus.

Embodiment B34. According to embodiment B33, wherein one of the one or more additional antigen binding domains at the N-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Single chain chimeric polypeptide.

Embodiment B35. a single-chain chimeric polypeptide 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; The single-chain chimeric polypeptide of embodiment B33, further comprising a linker sequence.

Embodiment B36. According to embodiment B33, wherein one of the one or more additional antigen binding domains at the C-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Single chain chimeric polypeptide.

Embodiment B37. a single chain chimeric polypeptide 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; The single-chain chimeric polypeptide of embodiment B33, further comprising a linker sequence.

Embodiment B38. Embodiments B26-B37, 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 IL-2 receptor or IL-15 receptor A single-chain chimeric polypeptide according to any one of

Embodiment B39. The single-chain chimeric polypeptide of embodiment B38, 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 B40. The single-chain chimeric polypeptide of any one of embodiments B26-B37, wherein the 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 a scFv or single domain antibody.

Embodiment B42. The one or more additional target binding domains are 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop -2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF -DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor , IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL -18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor , CD155 receptor, CD122 receptor, and CD28 receptor. Polypeptide.

Embodiment B43. The single-chain chimeric polypeptide of any one of embodiments B6-B37, B40, and B41, wherein the one or more additional target binding domains are soluble interleukin or cytokine proteins.

Embodiment B44. Soluble interleukin or cytokine protein is 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. The single-chain chimeric polypeptide of embodiment B43.

Embodiment B45. The single-chain chimeric polypeptide of any one of embodiments B6-B37, B40, and B41, wherein the 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 receptors are soluble TGF-beta receptor II (TGF-βRII) and soluble TGF-βRIII.

Embodiment B47. The single chain chimeric polypeptide of any one of embodiments B1-B46, wherein the single chain chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment B48. The single chain chimeric polypeptide according to any one of embodiments B1-B47, wherein the single chain chimeric polypeptide further comprises a peptide tag positioned at the N-terminus or C-terminus of the single chain chimeric polypeptide.

Embodiment B49. A composition comprising any of the single chain chimeric polypeptides of embodiments B1-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 of the single-chain chimeric polypeptides of any one of embodiments B1-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, comprising:
culturing the cells of embodiment B55 in a culture medium under conditions sufficient to result in the production of the single-chain chimeric polypeptide;
recovering the single 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 that is 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 that is at least 90% identical to SEQ ID NO:97.

Embodiment B60. The single-chain chimeric polypeptide of embodiment B59, wherein the soluble human tissue factor domain comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 100% identical to SEQ ID NO:98.

Embodiment C1. A multi-chain chimeric polypeptide,
(a) a first chimeric polypeptide,
(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,
a second chimeric polypeptide comprising (i) a second of the pair of affinity domains, and (ii) a second target binding domain;
the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of a pair of affinity domains;
Multi-chain chimeric polypeptides.

Embodiment C2. The multichain chimeric polypeptide of embodiment C1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide.

Embodiment C3. The multichain 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 within the first chimeric polypeptide.

Embodiment C4. The multichain chimeric polypeptide of any one of embodiments C1-C3, wherein the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. .

Embodiment C5. Any one of embodiments C1-C3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide A multi-chain chimeric polypeptide according to 1.

Embodiment C6. The multichain chimera of any one of embodiments C1-C5, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. Polypeptide.

Embodiment C7. Any of embodiments C1-C5, wherein the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide. or a multi-chain chimeric polypeptide according to any one of the above.

Embodiment C8. The multichain chimeric polypeptide of any one of embodiments C1-C7, wherein the first target binding domain and the second target binding domain specifically bind the same antigen.

Embodiment C9. The multichain 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 multichain 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 multichain chimeric polypeptide of any one of embodiments C1-C7, wherein the first target binding domain and the second target binding domain specifically bind different antigens.

Embodiment C12. The multi-chain chimeric polypeptide of any one of embodiments C1-C11, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

Embodiment C13. The multi-chain chimeric polypeptide of embodiment C12, wherein the first target binding domain and the second target binding domain are each antigen binding domains.

Embodiment C14. The multi-chain chimeric polypeptide of embodiment C12 or C13, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment C15. one or both of the first target binding domain and the second target binding domain are 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC , MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122 , CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL -1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 The multichain chimeric polypeptide of any one of embodiments C1-C14, which specifically binds a target selected from the group consisting of binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor. .

Embodiment C16. The multichain chimeric polypeptide of any one of embodiments C1-C14, wherein one or both of the first and second target binding domains is a soluble interleukin or cytokine protein.

Embodiment C17. Soluble interleukin or cytokine proteins are IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, PDGF-D, SCF, FLT3L, MICA, MICB, and ULP16 binding protein.

Embodiment C18. The multichain chimeric polypeptide of any one of embodiments C1-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 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 The multi-chain chimeric polypeptide of embodiment C18, which is

Embodiment C20. 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 comprises the soluble tissue factor domain and the first of the paired affinity domains. The multi-chain chimeric polypeptide of any one of embodiments C1-C19, which is positioned between a domain of

Embodiment C21. The first chimeric polypeptide 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 at least one of the one or more additional antigen binding domains. The multichain chimeric polypeptide of embodiment C20, further comprising a linker sequence between one and the first of the pair of affinity domains.

Embodiment C22. The multiple according to any one of embodiments C1-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. chain chimeric polypeptides.

Embodiment C23. According to embodiment C22, wherein at least one of the one or more additional target binding domains is directly adjacent to the first of the pair of affinity domains within the first chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment C24. The multiple 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 of the pair of affinity domains. chain chimeric polypeptides.

Embodiment C25. The multichain chimera of embodiment C22, wherein at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain within the first chimeric polypeptide Polypeptide.

Embodiment C26. The multichain 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. At least one of the one or more additional target binding domains is located at the N-terminus and/or C-terminus of the first chimeric polypeptide, wherein the one or more additional target binding domains The multichain chimeric polypeptide of embodiment C22, wherein at least one of is positioned between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide.

Embodiment C28. At least one additional target binding domain of the one or more additional target binding domains located at the N-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment C27, which is directly contiguous to one domain.

Embodiment C29. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment C27, further comprising a sequence.

Embodiment C30. At least one additional target binding domain of the one or more additional target binding domains located at the C-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment C27, which is directly contiguous to one domain.

Embodiment C31. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment C27, further comprising a sequence.

Embodiment C32. at least one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains is the soluble tissue factor domain and/or the affinity pair The multi-chain chimeric polypeptide of embodiment C27, wherein the domains are directly adjacent to the first domain.

Embodiment C33. the first chimeric polypeptide comprises: (i) the soluble tissue factor domain and one or more additional target binding domains positioned between the soluble tissue factor domain and the first of the pair of affinity domains; and/or (ii) between the first domain of the pair of affinity domains and the soluble tissue factor domain and the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment C27, further comprising a linker sequence interposed with at least one of the additional target binding domains of embodiment C27.

Embodiment C34. The multichain chimera of any one of embodiments C1-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. Polypeptide.

Embodiment C35. Described in embodiment C34, wherein at least one of the one or more additional target binding domains is directly adjacent to a second of the pair of affinity domains within the second chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment C36. 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 of the pair of affinity domains , embodiment C34.

Embodiment C37. The multichain chimera of embodiment C34, wherein at least one of the one or more additional target binding domains is directly adjacent to the second target binding domain within the second chimeric polypeptide Polypeptide.

Embodiment C38. 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 within the second chimeric polypeptide and the second target binding domain. 3. A multi-chain chimeric polypeptide according to .

Embodiment C39. Any one of embodiments C20-C38, 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. 3. A multi-chain chimeric polypeptide according to .

Embodiment C40. The multichain chimeric poly 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 the same epitope. peptide.

Embodiment C41. The multichain 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 multichain chimeric polypeptide of embodiment C39, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind the same antigen.

Embodiment C43. The multichain 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 the same epitope.

Embodiment C44. The multichain 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 multichain chimera of any one of embodiments C20-C38, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind different antigens. Polypeptide.

Embodiment C46. The multiple of any one of embodiments C20-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. chain chimeric polypeptides.

Embodiment C47. The multi-chain chimeric polypeptide of embodiment C46, wherein each of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is an antigen binding domain.

Embodiment C48. The multi-chain chimeric polypeptide of embodiment C47, wherein the antigen binding domain comprises a scFv.

Embodiment C49. one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA- DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand , scMHC I ligand, scMHC II ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor specifically binds to a target selected from the group consisting of receptors, MICA receptors, MICB receptors, ULP16 binding protein receptors, CD155 receptors, CD122 receptors, and CD3 receptors, and CD28 receptors. A multi-chain chimeric polypeptide according to any one of C20-C48.

Embodiment C50. Any one of embodiments C20-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 A multi-chain chimeric polypeptide according to 1.

Embodiment C51. soluble interleukin, cytokine, or ligand protein is 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 proteins.

Embodiment C52. Any of embodiments C20-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 1. A multi-chain chimeric polypeptide according to one.

Embodiment C53. 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, The multichain chimeric polypeptide of embodiment C52, which is soluble CD3, or soluble CD28.

Embodiment C54. The multi-chain chimeric polypeptide of any one of embodiments C1-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-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 multichain chimeric polypeptide of any one of embodiments C1-C55, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment C57. The multichain chimeric polypeptide of embodiment C56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93.

Embodiment C58. The multichain chimeric polypeptide of embodiment C57, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:93.

Embodiment C59. The multichain chimeric polypeptide of embodiment C58, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:93.

Embodiment C60. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method of any one of C56-C59.

Embodiment C61. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method described in C60.

Embodiment C62. The multichain chimeric polypeptide of any one of embodiments C1-C61, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment C63. The multichain chimeric polypeptide of any one of embodiments C1-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-C63, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment C65. The multichain chimeric polypeptide of any one of embodiments C1-C64, wherein the pair of affinity domains is a sushi domain derived from the alpha chain of the human IL-15 receptor (IL15Rα) 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 paired affinity domains are selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. A multi-chain chimeric polypeptide according to any one of embodiments C1-C64, wherein:

Embodiment C69. The multichain chimeric polypeptide according to any one of embodiments C1-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 multi-chain chimeric polypeptides of embodiments C1-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 of the multi-chain chimeric polypeptides of any one of embodiments C1-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 comprising:
culturing the cells of embodiment C76 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide;
recovering the multi-chain chimeric polypeptide from the cells and/or the culture medium.

Embodiment C78. A multi-chain chimeric polypeptide produced by the method of embodiment C77.

Embodiment C79. The multichain chimeric polypeptide of embodiment A56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:97.

Embodiment C80. The multichain chimeric polypeptide of embodiment C79, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:97.

Embodiment C81. The multichain chimeric polypeptide of embodiment C80, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:97.

Embodiment C82. The multichain chimeric polypeptide of embodiment C81, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:97.

Embodiment C83. The multichain chimeric polypeptide of embodiment C56, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:98.

Embodiment C84. The multichain chimeric polypeptide of embodiment C83, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:98.

Embodiment C85. The multichain chimeric polypeptide of embodiment C84, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:98.

Embodiment C86. The multichain chimeric polypeptide of embodiment C85, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:98.

Embodiment D1. A multi-chain chimeric polypeptide,
(a) a first chimeric polypeptide,
(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,
(i) a second domain of the pair of affinity domains, and (ii) a second target binding domain,
comprising a second chimeric polypeptide;
the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of a pair of affinity domains;
the first target binding domain and the second target binding domain each independently specifically bind to the IL-18 receptor or the IL-12 receptor;
Multi-chain chimeric polypeptides.

Embodiment D2. The multichain chimeric polypeptide of embodiment D1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide.

Embodiment D3. The multichain 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 within the first chimeric polypeptide.

Embodiment D4. The multichain chimeric polypeptide of any one of embodiments D1-D3, wherein the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. .

Embodiment D5. Any one of embodiments D1-D3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain within the first chimeric polypeptide and the first of the pair of affinity domains. A multi-chain chimeric polypeptide according to 1.

Embodiment D6. The multichain chimera of any one of embodiments D1-D5, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. Polypeptide.

Embodiment D7. Any of embodiments D1-D5, wherein the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide. or a multi-chain chimeric polypeptide according to any one of the preceding claims.

Embodiment D8. The multichain chimeric polypeptide of any one of embodiments D1-D7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment D9. The multichain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93.

Embodiment D10. The multichain chimeric polypeptide of embodiment D9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:93.

Embodiment D11. The multichain chimeric polypeptide of embodiment D10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:93.

Embodiment D12. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method according to any one of D8-D11.

Embodiment D13. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method of any one of D12.

Embodiment D14. The multichain chimeric polypeptide of any one of embodiments D1-D13, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment D15. The multichain chimeric polypeptide of any one of embodiments D1-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-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-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-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 according to any one of embodiments D1-D18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment D20. The multichain chimeric polypeptide of embodiment D19, wherein the signal sequence comprises SEQ ID NO:117.

Embodiment D21. The multichain chimeric polypeptide of embodiment D20, wherein the signal sequence is SEQ ID NO:117.

Embodiment D22. The multichain chimera of any one of embodiments D1-D21, wherein the pair of affinity domains is a sushi domain derived from the alpha chain of the human IL-15 receptor (IL-15Rα) and soluble IL-15 Polypeptide.

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 multichain chimeric polypeptide of embodiment D22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO:82.

Embodiment D25. The multichain chimeric polypeptide of embodiment D24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO:82.

Embodiment D26. The multichain chimeric polypeptide of embodiment D25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO:82.

Embodiment D27. The multichain 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-D27, wherein the sushi domain of IL-15Rα comprises a sushi domain derived from human IL-15Rα.

Embodiment D29. The multi-chain chimeric polypeptide of embodiment D28, wherein the sushi domain from human IL-15Rα comprises a sequence that is 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 that is 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 that is 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 derived from human IL-15Rα is mature full-length IL-15Rα.

Embodiment D34. The paired affinity domains are selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. A multi-chain chimeric polypeptide according to any one of embodiments D1-D21, wherein:

Embodiment D35. The multichain chimeric polypeptide of any one of embodiments D1-D34, wherein one or both of the first target binding domain and the second target binding domain are agonist antigen binding domains.

Embodiment D36. The multichain chimeric polypeptide of embodiment D35, wherein the first target binding domain and the second target binding domain are each agonist antigen binding domains.

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 multichain chimeric polypeptide of any one of embodiments D1-D34, wherein one or both of the first and second target binding domains is soluble IL-15 or soluble IL-18.

Embodiment D39. The multichain 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 multichain of any one of embodiments D1-D39, wherein both the first target binding domain and the second target binding domain specifically bind to IL-18 receptor or IL-12 receptor. chimeric polypeptide.

Embodiment D41. The multichain 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 multichain 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. Any one of embodiments D1-D39, wherein the first target binding domain specifically binds the IL-12 receptor and the second target binding domain specifically binds the IL-18 receptor multi-chain chimeric polypeptide of.

Embodiment D44. Any one of embodiments D1-D39, wherein the first target binding domain specifically binds the IL-18 receptor and the second target binding domain specifically binds the IL-12 receptor multi-chain chimeric polypeptide of.

Embodiment D45. The multichain chimeric polypeptide of embodiment D44, wherein the first target binding domain comprises soluble IL-18.

Embodiment D46. The multichain chimeric polypeptide of embodiment D45, wherein the soluble IL-18 is soluble human IL-18.

Embodiment D47. The multichain chimeric polypeptide of embodiment D46, wherein the soluble human IL-18 comprises a sequence that is at least 80% identical to SEQ ID NO:109.

Embodiment D48. The multichain chimeric polypeptide of embodiment D47, wherein the soluble human IL-18 comprises a sequence that is at least 90% identical to SEQ ID NO:109.

Embodiment D49. The multichain chimeric polypeptide of embodiment D48, wherein the soluble human IL-18 comprises a sequence that is at least 95% identical to SEQ ID NO:109.

Embodiment D50. The multichain chimeric polypeptide of embodiment D49, wherein the soluble human IL-18 comprises the sequence of SEQ ID NO:109.

Embodiment D51. The multichain chimeric polypeptide of any one of embodiments D44-D50, wherein the second target binding domain comprises soluble IL-12.

Embodiment D52. The multichain chimeric polypeptide of embodiment D51, wherein the soluble IL-18 is soluble human IL-12.

Embodiment D53. The multichain chimeric polypeptide of embodiment D52, wherein the soluble human IL-15 comprises a soluble human IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence.

Embodiment D54. The multichain chimeric polypeptide of embodiment D53, wherein the soluble human IL-15 further comprises a linker sequence between the soluble human IL-12β (p40) and soluble human IL-12α (p35) sequences.

Embodiment D55. The multichain chimeric polypeptide of embodiment D54, wherein the linker sequence comprises SEQ ID NO:102.

Embodiment D56. The multichain chimeric polypeptide of any one of embodiments D53-D55, wherein the soluble human IL-12β (p40) sequence comprises a sequence that is at least 80% identical to SEQ ID NO:81.

Embodiment D57. The multichain chimeric polypeptide of embodiment D56, wherein the soluble human IL-12β (p40) sequence comprises a sequence that is at least 90% identical to SEQ ID NO:81.

Embodiment D58. The multichain chimeric polypeptide of embodiment D57, wherein the soluble human IL-12β (p40) sequence comprises a sequence that is at least 95% identical to SEQ ID NO:81.

Embodiment D59. The multichain chimeric polypeptide of embodiment D58, wherein the soluble human IL-12β (p40) sequence comprises SEQ ID NO:81.

Embodiment D60. The multichain chimeric polypeptide of any one of embodiments D53-D59, wherein the soluble human IL-12α (p35) sequence comprises a sequence that is at least 80% identical to SEQ ID NO:80.

Embodiment D61. The multichain chimeric polypeptide of embodiment D60, wherein the soluble human IL-12α (p35) sequence comprises a sequence that is at least 90% identical to SEQ ID NO:80.

Embodiment D62. The mule chain chimeric polypeptide of embodiment D61, wherein the soluble human IL-12α (p35) sequence comprises a sequence that is at least 95% identical to SEQ ID NO:80.

Embodiment D63. The multichain chimeric polypeptide of embodiment D62, wherein the soluble human IL-12α (p35) sequence comprises SEQ ID NO:80.

Embodiment D64. The multi-chain chimeric polypeptide of embodiment D1, wherein the first chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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-D68, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 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 comprises the soluble tissue factor domain and the first of the paired affinity domains. A multi-chain chimeric polypeptide according to any one of embodiments D1-D63, which is positioned between a domain of

Embodiment D75. The first chimeric polypeptide 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 at least one of the one or more additional antigen binding domains. The multichain chimeric polypeptide of embodiment D74, further comprising a linker sequence between one and the first of the pair of affinity domains.

Embodiment D76. The multi-domain of any one of embodiments D1-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. chain chimeric polypeptides.

Embodiment D77. Described in embodiment D76, wherein at least one of the one or more additional target binding domains is directly adjacent to the first of the pair of affinity domains within the first chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment D78. The multiple 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 of the pair of affinity domains. chain chimeric polypeptides.

Embodiment D79. The multichain chimera of embodiment D76, wherein at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain within the first chimeric polypeptide Polypeptide.

Embodiment D80. The multichain 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.At least one of the one or more additional target binding domains is located at the N-terminus and/or C-terminus of the first chimeric polypeptide, wherein the one or more additional target binding domains The multichain chimeric polypeptide of embodiment D76, at least one of which is positioned between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide.

Embodiment D82. At least one additional target binding domain of the one or more additional target binding domains located at the N-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment D81, which is directly contiguous to one domain.

Embodiment D83. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment D81, further comprising a sequence.

Embodiment D84. At least one additional target binding domain of the one or more additional target binding domains located at the C-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment D81, which is directly contiguous to one domain.

Embodiment D85. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment D81, further comprising a sequence.

Embodiment D86. at least one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains is the soluble tissue factor domain and/or the affinity pair The multi-chain chimeric polypeptide of embodiment D81, wherein the domains are directly adjacent to the first domain.

Embodiment D87. the first chimeric polypeptide comprises: (i) the soluble tissue factor domain and one or more additional target binding domains positioned between the soluble tissue factor domain and the first of the pair of affinity domains; and/or (ii) between the first domain of the pair of affinity domains and the soluble tissue factor domain and the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment D81, further comprising a linker sequence interposed between and at least one of the additional target binding domains of embodiment D81.

Embodiment D88. Any one of embodiments D1-D63 and D74-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 multi-chain chimeric polypeptide of.

Embodiment D89. Described in embodiment D88, wherein at least one of the one or more additional target binding domains is directly adjacent to a second of the pair of affinity domains in the second chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment D90. 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 of the pair of affinity domains , embodiment D88.

Embodiment D91. The multichain chimera of embodiment D88, wherein at least one of the one or more additional target binding domains is directly adjacent to the second target binding domain within the second chimeric polypeptide Polypeptide.

Embodiment D92. 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 within the second chimeric polypeptide and the second target binding domain. 3. A multi-chain chimeric polypeptide according to .

Embodiment D93. Any one of embodiments D74-D92, 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 3. A multi-chain chimeric polypeptide according to .

Embodiment D94. The multichain chimeric poly 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 the same epitope. peptide.

Embodiment D95. The multichain chimeric polypeptide of embodiment B94, wherein two or more of the first target binding domain, second target binding domain, and one or more additional target binding domains comprise the same amino acid sequence.

Embodiment D96. The multichain chimera of any one of embodiments D74-D92, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind different antigens. Polypeptide.

Embodiment D97.The one or more additional antigen binding domains comprises 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop -2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF -DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor , IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL -18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor , CD155 receptor, and CD28 receptor.

Embodiment D98. The multichain chimeric polypeptide of any one of embodiments D74-D96, wherein the one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment D99. soluble interleukin, cytokine, or ligand protein is 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 protein.

Embodiment D100. The multichain chimeric polypeptide of any one of embodiments D74-D96, wherein the one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment D101. 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 the multichain chimeric polypeptide of embodiment B100, which is soluble CD28.

Embodiment D102. A composition comprising any of the multi-chain chimeric polypeptides of embodiments D1-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 of the multi-chain chimeric polypeptides of any one of embodiments D1-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 result in the production of the multi-chain chimeric polypeptide;
recovering the multi-chain chimeric polypeptide from the cells and/or the culture medium.

Embodiment D110. A multi-chain chimeric polypeptide produced by the method of embodiment D109.

Embodiment D111. The multichain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:97.

Embodiment D112. The multichain chimeric polypeptide of embodiment D111, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:97.

Embodiment D113. The multichain chimeric polypeptide of embodiment D112, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:97.

Embodiment D114. The multichain chimeric polypeptide of embodiment D113, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:97.

Embodiment D115. The multichain chimeric polypeptide of embodiment D8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:98.

Embodiment D116. The multichain chimeric polypeptide of embodiment D115, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:98.

Embodiment D117. The multichain chimeric polypeptide of embodiment D116, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:98.

Embodiment D118. The multichain chimeric polypeptide of embodiment D117, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:98.

Embodiment E1. A multi-chain chimeric polypeptide,
(a) a first chimeric polypeptide,
(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,
a second chimeric polypeptide comprising (i) a second of the pair of affinity domains, and (ii) a second target binding domain;
the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of a pair of affinity domains;
the first target binding domain and the second target binding domain each independently specifically bind to an IL-21 receptor or tumor growth factor receptor βII (TGFβRII) ligand;
Multi-chain chimeric polypeptides.

Embodiment E2. The multichain chimeric polypeptide of embodiment E1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide.

Embodiment E3. The multichain 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 within the first chimeric polypeptide.

Embodiment E4. The multichain chimeric polypeptide of any one of embodiments E1-E3, wherein the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. .

Embodiment E5. Any one of embodiments E1-E3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain within the first chimeric polypeptide and the first of the pair of affinity domains. A multi-chain chimeric polypeptide according to 1.

Embodiment E6. The multichain chimera of any one of embodiments E1-E5, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. Polypeptide.

Embodiment E7. Any of embodiments E1-E5, wherein the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide. or a multi-chain chimeric polypeptide according to any one of the above.

Embodiment E8. The multichain chimeric polypeptide of any one of embodiments E1-E7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment E9. The multichain chimeric polypeptide of embodiment E8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93.

Embodiment E10. The multichain chimeric polypeptide of embodiment E9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:93.

Embodiment E11. The multichain chimeric polypeptide of embodiment E10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:93.

Embodiment E12. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method of any one of E8-E11.

Embodiment E13. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. A method as described in E12.

Embodiment E14. The multichain chimeric polypeptide of any one of embodiments E1-E13, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment E15. The multichain chimeric polypeptide of any one of embodiments E1-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-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-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-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 according to any one of embodiments E1-E18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment E20. The multichain chimeric polypeptide of embodiment E19, wherein the signal sequence comprises SEQ ID NO:117.

Embodiment E21. The multichain chimeric polypeptide of embodiment E20, wherein the signal sequence is SEQ ID NO:117.

Embodiment E22. The multichain chimeric poly(polychain) of any one of embodiments E1-E21, wherein the pair of affinity domains is the alpha chain of the human IL-15 receptor (a sushi domain from IL-15Rα, and a soluble IL-15). peptide.

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 multichain chimeric polypeptide of embodiment E22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO:82.

Embodiment E25. The multichain chimeric polypeptide of embodiment E24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO:82.

Embodiment E26. The multichain chimeric polypeptide of embodiment E25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO:82.

Embodiment E27. The multichain 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-E27, wherein the sushi domain of IL-15Rα comprises a sushi domain derived 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 that is 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 that is 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 that is 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 derived from human IL-15Rα is mature full-length IL-15Rα.

Embodiment E34. The paired affinity domains are selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. The multi-chain chimeric polypeptide of any one of embodiments E1-E21, wherein

Embodiment E35. The multichain chimeric polypeptide of any one of embodiments E1-E34, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

Embodiment E36. The multichain 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 multichain chimeric polypeptide of any one of embodiments E1-E34, wherein one or both of the first and second target binding domains is soluble IL-21 or soluble TGFβRII.

Embodiment E39. The multichain chimeric polypeptide of any one of embodiments E1-E38, wherein both the first target binding domain and the second target binding domain specifically bind to IL-21 receptor or TGFβRII ligand. .

Embodiment E40. The multichain 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 multichain 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 multichain of any one of embodiments E1-E38, wherein the first target binding domain specifically binds a TGFβRII ligand and the second target binding domain specifically binds an IL-21 receptor. chimeric polypeptide.

Embodiment E43. The multichain of any one of embodiments E1-E38, wherein the first target binding domain specifically binds the IL-21 receptor and the second target binding domain specifically binds the TGFβRII ligand. chimeric polypeptide.

Embodiment E44. The multichain chimeric polypeptide of embodiment E43, wherein the first target binding domain comprises soluble IL-21.

Embodiment E45. The multichain chimeric polypeptide of embodiment E44, wherein the soluble IL-21 is soluble human IL-21.

Embodiment E46. The multichain 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 multichain 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 multichain chimeric polypeptide of embodiment E47, wherein the soluble human IL-21 comprises a sequence that is at least 95% identical to SEQ ID NO:83.

Embodiment E49. The multichain chimeric polypeptide of embodiment E48, wherein the soluble human IL-21 comprises the sequence of SEQ ID NO:83.

Embodiment E50. The multichain chimeric polypeptide of any one of embodiments E43-E49, wherein the second target binding domain comprises soluble TGFβRII.

Embodiment E51. The multichain chimeric polypeptide of embodiment E50, wherein the soluble TGFβRII is soluble human TGFβRII.

Embodiment E52. The multichain chimeric polypeptide of embodiment E51, wherein the soluble human TGFβRII comprises a first soluble human TGFβRII sequence and a second soluble human TGFβRII sequence.

Embodiment E53. The multichain chimeric polypeptide of embodiment E52, wherein the soluble human TGFβRII further comprises a linker sequence between the first soluble human TGFβRII sequence and the second soluble human TGFβRII sequence.

Embodiment E54. The multichain chimeric polypeptide of embodiment E53, wherein the linker sequence comprises SEQ ID NO:102.

Embodiment E55. The multichain chimeric polypeptide of any one of embodiments E52-E54, wherein the first soluble human TGFβRII sequence comprises a sequence that is at least 80% identical to SEQ ID NO:183.

Embodiment E56. The multichain chimeric polypeptide of embodiment E55, wherein the first soluble human TGFβRII sequence comprises a sequence that is at least 90% identical to SEQ ID NO:183.

Embodiment E57. The multichain chimeric polypeptide of embodiment E56, wherein the first soluble human TGFβRII sequence comprises a sequence that is at least 95% identical to SEQ ID NO:183.

Embodiment E58. The multichain chimeric polypeptide of embodiment E57, wherein the first soluble human TGFβRII sequence comprises SEQ ID NO:183.

Embodiment E59. The multichain chimeric polypeptide of any one of embodiments E52-E58, wherein the second soluble human TGFβRII sequence comprises a sequence that is at least 80% identical to SEQ ID NO:184.

Embodiment E60. The multichain chimeric polypeptide of embodiment E59, wherein the second soluble human TGFβRII sequence comprises a sequence that is at least 90% identical to SEQ ID NO:184.

Embodiment E61. The mule chain chimeric polypeptide of embodiment E60, wherein the second soluble human TGFβRII sequence comprises a sequence that is at least 95% identical to SEQ ID NO:184.

Embodiment E62. The multichain chimeric polypeptide of embodiment E61, wherein the second soluble human TGFβRII sequence comprises SEQ ID NO:184.

Embodiment E63. The multi-chain chimeric polypeptide of embodiment E1, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:189.

Embodiment E64. The multichain chimeric polypeptide of embodiment E63, wherein the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:189.

Embodiment E65. The multichain chimeric polypeptide of embodiment E64, wherein the first chimeric polypeptide comprises a sequence that is 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-E67, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:193.

Embodiment E69. The multichain chimeric polypeptide of embodiment E68, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 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 comprises the soluble tissue factor domain and the first of the paired affinity domains. A multi-chain chimeric polypeptide according to any one of embodiments E1-E62, which is positioned between a domain of

Embodiment E74. The first chimeric polypeptide 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 at least one of the one or more additional antigen binding domains. The multichain chimeric polypeptide of embodiment E73, further comprising a linker sequence between one and the first of the pair of affinity domains.

Embodiment E75. The multiple according to any one of embodiments E1-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. chain chimeric polypeptides.

Embodiment E76. Described in embodiment E75, wherein at least one of the one or more additional target binding domains is directly adjacent to the first of the pair of affinity domains within the first chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment E77. The multiple 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 of the pair of affinity domains. chain chimeric polypeptides.

Embodiment E78. The multichain chimera of embodiment E75, wherein at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain within the first chimeric polypeptide Polypeptide.

Embodiment E79. The multichain 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. At least one of the one or more additional target binding domains is located at the N-terminus and/or C-terminus of the first chimeric polypeptide, wherein the one or more additional target binding domains are The multichain chimeric polypeptide of embodiment E75, wherein at least one of is positioned between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide.

Embodiment E81. At least one additional target binding domain of the one or more additional target binding domains located at the N-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment E80, which is directly contiguous to one domain.

Embodiment E82. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment E80, further comprising a sequence.

Embodiment E83. At least one additional target binding domain of the one or more additional target binding domains located at the C-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment E80, which is directly contiguous to one domain.

Embodiment E84. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment E80, further comprising a sequence.

Embodiment E85. at least one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains is the soluble tissue factor domain and/or the affinity pair The multi-chain chimeric polypeptide of embodiment E80, wherein the domains are directly adjacent to the first domain.

Embodiment E86. the first chimeric polypeptide comprises: (i) the soluble tissue factor domain and one or more additional target binding domains positioned between the soluble tissue factor domain and the first of the pair of affinity domains; and/or (ii) between the first domain of the pair of affinity domains and the soluble tissue factor domain and the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment E80, further comprising a linker sequence interposed with at least one of the additional target binding domains of embodiment E80.

Embodiment E87. Any one of embodiments E1-E62 and E73-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 multi-chain chimeric polypeptide of.

Embodiment E88. Described in embodiment E87, wherein at least one of the one or more additional target binding domains is directly adjacent to a second of the pair of affinity domains within the second chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment E89. 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 of the pair of affinity domains , embodiment E87.

Embodiment E90. The multichain chimera of embodiment E87, wherein at least one of the one or more additional target binding domains is directly adjacent to the second target binding domain within the second chimeric polypeptide Polypeptide.

Embodiment E91. 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 within the second chimeric polypeptide and the second target binding domain. 3. A multi-chain chimeric polypeptide according to .

Embodiment E92. Any one of embodiments E73-E91, 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 3. A multi-chain chimeric polypeptide according to .

Embodiment E93. The multichain chimeric poly 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 the same epitope. peptide.

Embodiment E94. The multichain chimeric polypeptide of embodiment E93, wherein two or more of the first target binding domain, second target binding domain, and one or more additional target binding domains comprise the same amino acid sequence.

Embodiment E95. The multichain chimera of any one of embodiments E73-E91, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind different antigens. Polypeptide.

Embodiment E96.The one or more additional antigen binding domains comprises 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop -2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF -D, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor , IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL -18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor , CD155 receptor, and CD28 receptor.

Embodiment E97. The multichain chimeric polypeptide of any one of embodiments E73-E95, wherein the one or more additional target binding domains is a soluble interleukin or cytokine protein.

Embodiment E98. soluble interleukin, cytokine, or ligand protein is 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 protein.

Embodiment E99. The multichain chimeric polypeptide of any one of embodiments E73-E95, wherein the one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment E100. 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 The multi-chain chimeric polypeptide of embodiment E99, which is

Embodiment E101. A composition comprising any of the multi-chain chimeric polypeptides of embodiments E1-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 of the multi-chain chimeric polypeptides of any one of embodiments E1-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 comprising:
culturing the cells of embodiment E107 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide;
recovering the multi-chain chimeric polypeptide from the cells and/or the culture medium.

Embodiment E109. A multi-chain chimeric polypeptide produced by the method of embodiment E108.

Embodiment E110. The multichain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:97.

Embodiment E111. The multichain chimeric polypeptide of embodiment E110, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:97.

Embodiment E112. The multichain chimeric polypeptide of embodiment E111, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:97.

Embodiment E113. The multichain chimeric polypeptide of embodiment E112, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:97.

Embodiment E114. The multichain chimeric polypeptide of embodiment E12, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:98.

Embodiment E115. The multichain chimeric polypeptide of embodiment E114, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:98.

Embodiment E116. The multichain chimeric polypeptide of embodiment E115, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:98.

Embodiment E117. The multichain chimeric polypeptide of embodiment E116, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:98.

Embodiment F1. A multi-chain chimeric polypeptide,
(c) a first chimeric polypeptide,
(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,
a second chimeric polypeptide comprising (i) a second of the pair of affinity domains, and (ii) a second target binding domain;
the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of a pair of affinity domains;
the first target binding domain and the second target binding domain each independently specifically bind to the IL-21 receptor or the IL-7 receptor;
Multi-chain chimeric polypeptides.

Embodiment F2. The multichain chimeric polypeptide of embodiment F1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide.

Embodiment F3. The multichain 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 within the first chimeric polypeptide.

Embodiment F4. The multichain chimeric polypeptide of any one of embodiments F1-F3, wherein the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. .

Embodiment F5. Any one of embodiments F1-F3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. A multi-chain chimeric polypeptide according to 1.

Embodiment F6. The multichain chimera of any one of embodiments F1-F5, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. Polypeptide.

Embodiment F7. Any of embodiments F1-F5, wherein the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide. or a multi-chain chimeric polypeptide according to any one of the above.

Embodiment F8. The multichain chimeric polypeptide of any one of embodiments F1-F7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment F9. The multichain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93.

Embodiment F10. The multichain chimeric polypeptide of embodiment F9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:93.

Embodiment F11. The multichain chimeric polypeptide of embodiment F10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:93.

Embodiment F12. The multichain chimeric polypeptide of embodiment F11, wherein the soluble human tissue factor domain comprises SEQ ID NO:93.

Embodiment F13. The multichain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:97.

Embodiment F14. The multichain chimeric polypeptide of embodiment F13, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:97.

Embodiment F15. The multichain chimeric polypeptide of embodiment F14, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:97.

Embodiment F16. The multichain chimeric polypeptide of embodiment F15, wherein the soluble human tissue factor domain comprises SEQ ID NO:97.

Embodiment F17. The multichain chimeric polypeptide of embodiment F8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:98.

Embodiment F18. The multichain chimeric polypeptide of embodiment F17, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:98.

Embodiment F19. The multichain chimeric polypeptide of embodiment F18, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:98.

Embodiment F20. The multichain chimeric polypeptide of embodiment F19, wherein the soluble human tissue factor domain comprises SEQ ID NO:98.

Embodiment F21. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method of any one of F8-F11, F13-F15, and F17-F19.

Embodiment F22. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method described in F21.

Embodiment F23. The multichain chimeric polypeptide of any one of embodiments F1-F22, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment F24. The multichain chimeric polypeptide of any one of embodiments F1-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-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-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-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 according to any one of embodiments F1-F27, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment F29. The multichain chimeric polypeptide of embodiment F28, wherein the signal sequence comprises SEQ ID NO:117.

Embodiment F30. The multichain chimeric polypeptide of embodiment F28, wherein the signal sequence is SEQ ID NO:328.

Embodiment F31. The multi-chain chimera of any one of embodiments F1-F30, wherein the pair of affinity domains is a sushi domain derived from the alpha chain of the human IL-15 receptor (IL-15Rα) and soluble IL-15. Polypeptide.

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 multichain chimeric polypeptide of embodiment F31, wherein the soluble IL-15 comprises a sequence that is at least 80% identical to SEQ ID NO:82.

Embodiment F34. The multichain chimeric polypeptide of embodiment F33, wherein the soluble IL-15 comprises a sequence that is at least 90% identical to SEQ ID NO:82.

Embodiment F35. The multichain chimeric polypeptide of embodiment F34, wherein the soluble IL-15 comprises a sequence that is at least 95% identical to SEQ ID NO:82.

Embodiment F36. The multichain 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-F36, wherein the sushi domain of IL-15Rα comprises a sushi domain derived from human IL-15Rα.

Embodiment F38. The multi-chain chimeric polypeptide of embodiment F37, wherein the sushi domain from human IL-15Rα comprises a sequence that is 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 that is 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 that is 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 derived from human IL-15Rα is mature full-length IL-15Rα.

Embodiment F43. The paired affinity domains are selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. A multi-chain chimeric polypeptide according to any one of embodiments F1-F30, wherein:

Embodiment F44. The multichain chimeric polypeptide of any one of embodiments F1-F43, wherein one or both of the first target binding domain and the second target binding domain are agonist antigen binding domains.

Embodiment F45. The multi-chain chimeric polypeptide of embodiment F44, wherein the first target binding domain and the second target binding domain are each agonist antigen binding domains.

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 multichain chimeric polypeptide of any one of embodiments F1-F43, wherein one or both of the first and second target binding domains is soluble IL-21 or soluble IL-7.

Embodiment F48. The multichain 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 multichain of any one of embodiments F1-F48, wherein both the first target binding domain and the second target binding domain specifically bind to IL-21 receptor or IL-7 receptor. chimeric polypeptide.

Embodiment F50. The multichain 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. Any one of embodiments F1-F48, wherein the first target binding domain specifically binds the IL-21 receptor and the second target binding domain specifically binds the IL-7 receptor. multi-chain chimeric polypeptide of.

Embodiment F53. Any one of embodiments F1-F48, wherein the first target binding domain specifically binds the IL-7 receptor and the second target binding domain specifically binds the IL-21 receptor. multi-chain chimeric polypeptide of.

Embodiment F54. The multichain chimeric polypeptide of embodiment F53, wherein the first target binding domain comprises soluble IL-21.

Embodiment F55. The multichain chimeric polypeptide of embodiment F54, wherein the soluble IL-21 is soluble human IL-21.

Embodiment F56. The multichain 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 multichain 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 multichain 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 multichain chimeric polypeptide of embodiment F58, wherein the soluble human IL-21 comprises the sequence of SEQ ID NO:83.

Embodiment F60. The multichain chimeric polypeptide of any one of embodiments F53-F59, wherein the second target binding domain comprises soluble IL-7.

Embodiment F61. The multichain chimeric polypeptide of embodiment D60, wherein the soluble IL-7 is soluble human IL-7.

Embodiment F62. The multichain chimeric polypeptide of embodiment F61, wherein the soluble human IL-7 comprises a sequence that is at least 80% identical to SEQ ID NO:79.

Embodiment F63. The multichain 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 multichain chimeric polypeptide of embodiment F63, wherein the soluble human IL-7 comprises a sequence that is at least 95% identical to SEQ ID NO:79.

Embodiment F65. The multichain 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 that is 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 that is 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 that is at least 95% identical to SEQ ID NO:207.

Embodiment F69. A multi-chain chimeric polypeptide according to 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-F70, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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-E80, wherein the second chimeric polypeptide comprises a sequence that is at least 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 that is 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 that is 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.

Embodiment F86. 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 comprises the soluble tissue factor domain and the first of the paired affinity domains. The multi-chain chimeric polypeptide of any one of embodiments F1-F65, wherein the multi-chain chimeric polypeptide of any one of embodiments F1-F65 is positioned between a domain of

Embodiment F87. The first chimeric polypeptide 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 at least one of the one or more additional antigen binding domains. The multi-chain chimeric polypeptide of embodiment F86, further comprising a linker sequence between one and the first of the pair of affinity domains.

Embodiment F88. The multiple according to any one of embodiments F1-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. chain chimeric polypeptides.

Embodiment F89. According to embodiment F88, wherein at least one of the one or more additional target binding domains is directly adjacent to the first of the pair of affinity domains within the first chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment F90. The multiple 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 of the pair of affinity domains. chain chimeric polypeptides.

Embodiment F91. The multichain chimera of embodiment F88, wherein at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain within the first chimeric polypeptide Polypeptide.

Embodiment F92. The multichain 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. At least one of the one or more additional target binding domains is located at the N-terminus and/or C-terminus of the first chimeric polypeptide, wherein the one or more additional target binding domains The multichain chimeric polypeptide of embodiment F88, wherein at least one of is positioned between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide.

Embodiment F94. At least one additional target binding domain of the one or more additional target binding domains located at the N-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains A multi-chain chimeric polypeptide according to embodiment F93, which is directly contiguous to one domain.

Embodiment F95. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment F93, further comprising a sequence.

Embodiment F96. At least one additional target binding domain of the one or more additional target binding domains located at the C-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains A multi-chain chimeric polypeptide according to embodiment F93, which is directly contiguous to one domain.

Embodiment F97. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment F93, further comprising a sequence.

Embodiment F98. at least one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains is the soluble tissue factor domain and/or the affinity pair The multi-chain chimeric polypeptide of embodiment F93, wherein the domains are directly adjacent to the first domain.

Embodiment F99. the first chimeric polypeptide comprises: (i) the soluble tissue factor domain and one or more additional target binding domains positioned between the soluble tissue factor domain and the first of the pair of affinity domains; and/or (ii) between the first domain of the pair of affinity domains and the soluble tissue factor domain and the first domain of the pair of affinity domains The multi-chain chimeric polypeptide of embodiment F93, further comprising a linker sequence interposed with at least one of the additional target binding domains of embodiment F93.

Embodiment F100. Any one of embodiments F1-F65 and F86-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 multi-chain chimeric polypeptide of.

Embodiment F101. According to embodiment F100, wherein at least one of the one or more additional target binding domains is directly adjacent to a second domain of the pair of affinity domains within the second chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment F102. 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 of the pair of affinity domains , embodiment F100.

Embodiment F103. The multichain chimera of embodiment F100, wherein at least one of the one or more additional target binding domains is directly adjacent to the second target binding domain within the second chimeric polypeptide. Polypeptide.

Embodiment F104. 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 within the second chimeric polypeptide and the second target binding domain. 3. A multi-chain chimeric polypeptide according to .

Embodiment F105. Any one of embodiments F86-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. 3. A multi-chain chimeric polypeptide according to .

Embodiment F106. The multichain chimeric poly 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 the same epitope. peptide.

Embodiment F107. The multichain chimeric polypeptide of embodiment F106, wherein two or more of the first target binding domain, second target binding domain, and one or more additional target binding domains comprise the same amino acid sequence.

Embodiment F108. The multichain chimera of any one of embodiments F86-F104, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind different antigens. Polypeptide.

Embodiment F109. The one or more additional antigen binding domains comprises 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, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop -2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF -DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor , IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL -18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor , CD155 receptor, and CD28 receptor.

Embodiment F110. The multichain chimeric polypeptide of any one of embodiments F86-F108, wherein the one or more additional target binding domains are soluble interleukin or cytokine proteins.

Embodiment F111. soluble interleukin, cytokine, or ligand protein is 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 protein.

Embodiment F112. The multichain chimeric polypeptide of any one of embodiments F86-F108, wherein the one or more additional target binding domains is a soluble interleukin or cytokine receptor.

Embodiment F113. 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 the multichain chimeric polypeptide of embodiment F112, which is soluble CD28.

Embodiment F114. A composition comprising any of the multi-chain chimeric polypeptides of embodiments F1-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 of the multi-chain chimeric polypeptides of any one of embodiments F1-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 comprising:
culturing the cells of embodiment F120 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide;
recovering the multi-chain chimeric polypeptide from the cells and/or the culture medium.

Embodiment F122. A multi-chain chimeric polypeptide produced by the method of embodiment F121.

Embodiment G1. A multi-chain chimeric polypeptide,
(e) a first chimeric polypeptide,
(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,
a second chimeric polypeptide comprising (i) a second of the pair of affinity domains, and (ii) a second target binding domain;
the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of a pair of affinity domains;
the first target binding domain and the second target binding domain each independently specifically bind to IL-7 receptor, CD16, IL-21 receptor, TGF-β, or CD137L receptor;
Multi-chain chimeric polypeptides.

Embodiment G2. The multichain chimeric polypeptide of embodiment G1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide.

Embodiment G3. The multichain 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 within the first chimeric polypeptide.

Embodiment G4. The multichain chimeric polypeptide of any one of embodiments G1-G3, wherein the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide. .

Embodiment G5. Any one of embodiments G1-G3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain within the first chimeric polypeptide and the first of the pair of affinity domains. A multi-chain chimeric polypeptide according to 1.

Embodiment G6. The multichain chimera of any one of embodiments G1-G5, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide. Polypeptide.

Embodiment G7. Any of embodiments G1-G5, wherein the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide. or a multi-chain chimeric polypeptide according to any one of the above.

Embodiment G8. The multichain chimeric polypeptide of any one of embodiments G1-G7, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment G9. The multichain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93.

Embodiment G10. The multichain chimeric polypeptide of embodiment G9, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:93.

Embodiment G11. The multichain chimeric polypeptide of embodiment G10, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:93.

Embodiment G12. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method according to any one of G8-G11.

Embodiment G13. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method described in G12.

Embodiment G14. The multichain chimeric polypeptide of any one of embodiments G1-G13, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment G15. The multichain chimeric polypeptide of any one of embodiments G1-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-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-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-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 according to any one of embodiments G1-G18, wherein the first chimeric polypeptide and/or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

Embodiment G20. The multichain chimeric polypeptide of embodiment G19, wherein the signal sequence comprises SEQ ID NO:117.

Embodiment G21. The multichain chimeric polypeptide of embodiment G20, wherein the signal sequence is SEQ ID NO:117.

Embodiment G22. The multichain chimera of any one of embodiments G1-G21, wherein the paired affinity domains are a sushi domain derived from the alpha chain of the human IL-15 receptor (IL-15Rα) and soluble IL-15. Polypeptide.

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 multichain chimeric polypeptide of embodiment G22, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO:82.

Embodiment G25. The multichain chimeric polypeptide of embodiment G24, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO:82.

Embodiment G26. The multichain chimeric polypeptide of embodiment G25, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO:82.

Embodiment G27. The multichain 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-G27, wherein the sushi domain of IL-15Rα comprises a sushi domain derived from human IL-15Rα.

Embodiment G29. The multi-chain chimeric polypeptide of embodiment G28, wherein the sushi domain from human IL-15Rα comprises a sequence that is 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 that is 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 that is 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 derived from human IL-15Rα is mature full-length IL-15Rα.

Embodiment G34. The paired affinity domains are selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. The multi-chain chimeric polypeptide of any one of embodiments G1-G21, wherein:

Embodiment G35. Any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to IL-7 receptor, CD16, or IL-21 receptor 3. A multi-chain chimeric polypeptide according to .

Embodiment G36. The multichain chimera of embodiment G35, wherein the first target binding domain specifically binds the IL-7 receptor and the second target binding domain specifically binds the CD16 or IL-21 receptor. Polypeptide.

Embodiment G37. The multichain chimeric polypeptide of embodiment G36, wherein the first target binding domain comprises a soluble IL-7 protein.

Embodiment G38. The multichain chimeric polypeptide of embodiment G37, wherein the soluble IL-7 protein is soluble human IL-7.

Embodiment G39. The multichain chimeric polypeptide of any one of embodiments G36-G38, wherein the second antigen binding domain comprises an antigen binding domain that specifically binds CD16.

Embodiment G40. The multichain chimeric polypeptide of embodiment G39, wherein the second antigen binding domain comprises an scFv that specifically binds CD16.

Embodiment G41. The multi-chain chimeric polypeptide of any one of embodiments G36-G38, wherein the second antigen binding domain specifically binds the IL-21 receptor.

Embodiment G42. The multichain chimeric polypeptide of embodiment G41, wherein the second antigen binding domain comprises soluble IL-21.

Embodiment G43. The multichain chimeric polypeptide of embodiment G42, wherein the soluble IL-21 is soluble human IL-21.

Embodiment G44. The multichain chimeric polypeptide of any one of embodiments G36-G40, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds the IL-21 receptor.

Embodiment G45. The multichain chimeric polypeptide of embodiment G44, wherein the additional target binding domain comprises soluble IL-21.

Embodiment G46. The multichain chimeric polypeptide of embodiment G45, wherein the soluble IL-21 is soluble human IL-12.

Embodiment G47. Any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to a TGF-beta, CD16, or IL-21 receptor. multi-chain chimeric polypeptide of.

Embodiment G48. The multichain chimeric polypeptide of embodiment G47, wherein the first target binding domain specifically binds TGF-beta and the second target binding domain specifically binds the CD16 or IL-21 receptor. .

Embodiment G49. The multispecific chimeric polypeptide of embodiment G48, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G50. The multispecific chimeric polypeptide of embodiment G49, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G51. The multispecific chimeric polypeptide of any one of embodiments G48-G50, wherein the second target binding domain specifically binds CD16.

Embodiment G52. The multispecific chimeric polypeptide of embodiment G51, wherein the second antigen binding domain comprises an antigen binding domain that specifically binds CD16.

Embodiment G53. The multichain chimeric polypeptide of embodiment G52, wherein the second antigen binding domain comprises an scFv that specifically binds CD16.

Embodiment G54. The multichain chimeric polypeptide of any one of embodiments G48-G50, wherein the second target binding domain specifically binds to the IL-21 receptor.

Embodiment G55. The multichain chimeric polypeptide of embodiment G54, wherein the second target binding domain comprises soluble IL-21.

Embodiment G56. The multichain chimeric polypeptide of embodiment G55, wherein the second target binding domain comprises soluble human IL-21.

Embodiment G57. The multichain chimeric polypeptide of any one of embodiments G48-G53, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds the IL-21 receptor.

Embodiment G58. The multichain chimeric polypeptide of embodiment G57, wherein the additional target binding domain comprises soluble IL-21.

Embodiment G59. The multichain chimeric polypeptide of embodiment G58, wherein the soluble IL-21 is soluble human IL-21.

Embodiment G60. The multichain chimeric polypeptide of any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to IL-7 receptor.

Embodiment G61. The multichain chimeric polypeptide of embodiment G60, wherein the first target binding domain and the second target binding domain comprise soluble IL-7.

Embodiment G62. The multichain chimeric polypeptide of embodiment G61, wherein the soluble IL-7 is soluble human IL-7.

Embodiment G63. The multichain chimeric polypeptide of any one of embodiments G1-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-beta receptors.

Embodiment G65. The multispecific chimeric polypeptide of embodiment G64, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G66. Any of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to IL-7 receptor, IL-21 receptor, or CD137L receptor 1. A multispecific chimeric polypeptide according to one.

Embodiment G67. The multichain of embodiment G66, wherein the first target binding domain specifically binds IL-7 receptor and the second target binding domain specifically binds IL-21 receptor or CD137L receptor. chimeric polypeptide.

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 multichain chimeric polypeptide of any one of embodiments G67-G69, wherein the second target binding domain specifically binds the IL-21 receptor.

Embodiment G71. The multichain chimeric polypeptide of embodiment G70, wherein the second target binding domain is soluble IL-21.

Embodiment G72. The multichain chimeric polypeptide of embodiment G71, wherein the soluble IL-21 is soluble human IL-21.

Embodiment G73. The multichain chimeric polypeptide of any one of embodiments G67-G69, wherein the second antigen binding domain specifically binds to the CD137L receptor.

Embodiment G74. The multichain chimeric polypeptide of embodiment G73, wherein the second antigen binding domain is soluble CD137L.

Embodiment G75. The multichain chimeric polypeptide of embodiment G74, wherein the soluble CD137L is soluble human CD137L.

Embodiment G76. The multichain chimeric polypeptide of any one of embodiments G67-G72, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to the CD137L receptor.

Embodiment G77. The multichain chimeric polypeptide of embodiment G76, wherein the additional target binding domain comprises soluble CD137L.

Embodiment G78. The multichain chimeric polypeptide of embodiment G77, wherein the soluble CD137L is soluble human CD137L.

Embodiment G79. The multichain of any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to IL-7 receptor or TGF-β. chimeric polypeptide.

Embodiment G80. The multichain chimeric polypeptide of embodiment G79, wherein the first target binding domain specifically binds IL-7 receptor and the second target binding domain specifically binds TGF-β.

Embodiment G81. The multichain chimeric polypeptide of embodiment G80, wherein the first target binding domain comprises a soluble IL-7 protein.

Embodiment G82. The multichain chimeric polypeptide of embodiment G81, wherein the soluble IL-7 protein is soluble human IL-7.

Embodiment G83. The multichain chimeric polypeptide of any one of embodiments G80-G82, wherein the second antigen binding domain comprises an antigen binding domain that specifically binds TGF-β.

Embodiment G84. The multispecific chimeric polypeptide of embodiment G83, wherein the second target binding domain is a soluble TGF-beta receptor.

Embodiment G85. The multispecific chimeric polypeptide of embodiment G84, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G86. Any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to TGF-β, IL-21 receptor, or CD137L receptor 3. A multi-chain chimeric polypeptide according to .

Embodiment G87. The multichain chimeric poly of embodiment G86, wherein the first target binding domain specifically binds TGF-β and the second target binding domain specifically binds IL-21 receptor or CD137L receptor. peptide.

Embodiment G88. The multispecific chimeric polypeptide of embodiment G87, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G89. The multispecific chimeric polypeptide of embodiment G88, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G90. The multispecific chimeric polypeptide of any one of embodiments G87-G89, wherein the second target binding domain specifically binds the IL-21 receptor.

Embodiment G91. The multichain chimeric polypeptide of embodiment G90, wherein the second target binding domain comprises soluble IL-21.

Embodiment G92. The multichain 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-G89, wherein the second target binding domain specifically binds to the CD137L receptor.

Embodiment G94. The multichain chimeric polypeptide of embodiment G93, wherein the second target binding domain comprises soluble CD137L.

Embodiment G95. The multichain chimeric polypeptide of embodiment G94, wherein the second target binding domain comprises soluble human CD137L.

Embodiment G96. The multichain chimeric polypeptide of any one of embodiments G87-G92, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to the CD137L receptor.

Embodiment G97. The multichain chimeric polypeptide of embodiment G96, wherein the additional target binding domain comprises soluble CD137L.

Embodiment G98. The multichain chimeric polypeptide of embodiment G97, wherein the soluble CD137L is soluble human CD137L.

Embodiment G99. The multichain of any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to TGF-β or IL-21 receptor. chimeric polypeptide.

Embodiment G100. The multichain chimeric poly of embodiment G99, wherein the first target binding domain specifically binds TGF-beta and the second target binding domain specifically binds TGF-beta or IL-21 receptor. peptide.

Embodiment G101. The multispecific chimeric polypeptide of embodiment G100, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G102. The multispecific chimeric polypeptide of embodiment G101, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G103. The multispecific chimeric polypeptide of any one of embodiments G100-G102, wherein the second target binding domain specifically binds the IL-21 receptor.

Embodiment G104. The multichain chimeric polypeptide of embodiment G103, wherein the second target binding domain comprises soluble IL-21.

Embodiment G105. The multichain 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-G102, wherein the second target binding domain specifically binds TGF-β.

Embodiment G107. The multispecific chimeric polypeptide of embodiment G106, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G108. The multispecific chimeric polypeptide of embodiment G107, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G109. The multispecific chimeric polypeptide of any one of embodiments G100-G105, wherein the second polypeptide further comprises an additional target binding domain that specifically binds TGF-β.

Embodiment G110. The multispecific chimeric polypeptide of embodiment G109, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G111. The multispecific chimeric polypeptide of embodiment G110, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G112. The multichain chimeric poly of any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to TGF-β or IL-16. peptide.

Embodiment G113. The multichain chimeric polypeptide of embodiment G112, wherein the first target binding domain specifically binds TGF-beta and the second target binding domain specifically binds TGF-beta or IL-16.

Embodiment G114. The multispecific chimeric polypeptide of embodiment G113, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G115. The multispecific chimeric polypeptide of embodiment G114, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G116. The multispecific chimeric polypeptide of any one of embodiments G113-G115, wherein the second target binding domain specifically binds 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 CD16.

Embodiment G118. The multichain chimeric polypeptide of embodiment G117, wherein the second antigen binding domain comprises an scFv that specifically binds CD16.

Embodiment G119. The multispecific chimeric polypeptide of any one of embodiments G113-G115, wherein the second target binding domain specifically binds TGF-β.

Embodiment G120. The multispecific chimeric polypeptide of embodiment G119, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G121. The multispecific chimeric polypeptide of embodiment G120, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G122. The multispecific chimeric polypeptide of any one of embodiments G113-G118, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds TGF-β.

Embodiment G123. The multispecific chimeric polypeptide of embodiment G122, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G124. The multispecific chimeric polypeptide of embodiment G123, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G125. The multichain chimeric poly of any one of embodiments G1-G34, wherein the first target binding domain and the second target binding domain each independently specifically bind to TGF-β or CD137L receptor. peptide.

Embodiment G126. The multichain chimeric polypeptide of embodiment G125, wherein the first target binding domain specifically binds TGF-β and the second target binding domain specifically binds the CD137L receptor.

Embodiment G127. The multispecific chimeric polypeptide of embodiment G126, wherein the first target binding domain is a soluble TGF-beta receptor.

Embodiment G128. The multispecific chimeric polypeptide of embodiment G127, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G129. The multichain chimeric polypeptide of embodiment G128, wherein the second target binding domain comprises a soluble CD137L protein.

Embodiment G130. The multichain chimeric polypeptide of embodiment G129, wherein the soluble CD137L protein is soluble human CD137L.

Embodiment G131. The multichain chimeric polypeptide of any one of embodiments G126-G130, wherein the second chimeric polypeptide further comprises an additional target binding domain that specifically binds TGF-β.

Embodiment G132. The multispecific chimeric polypeptide of embodiment G131, wherein the additional target binding domain is a soluble TGF-beta receptor.

Embodiment G133. The multispecific chimeric polypeptide of embodiment G132, wherein the soluble TGF-beta receptor is a soluble TGFbetaRII receptor.

Embodiment G134. The multi-chain chimeric polypeptide of embodiment G1, wherein the first chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G134-G138, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G144-G148, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:232.

Embodiment G150. The multichain chimeric polypeptide of embodiment G149, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G154-G158, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:203.

Embodiment G160. The multichain chimeric polypeptide of embodiment G159, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G164-G168, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:193.

Embodiment G170. The multichain chimeric polypeptide of embodiment G169, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G174-G178, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G184-G188, wherein the second chimeric polypeptide comprises a sequence that is 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 that is at least 90% identical to SEQ ID NO:272.

Embodiment G191. The multichain chimeric polypeptide of embodiment G190, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G194-G198, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:193.

Embodiment G200. The multichain chimeric polypeptide of embodiment G199, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:193.

Embodiment G201. The multichain chimeric polypeptide of embodiment G200, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G204-G208, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G214-G218, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G224-G228, wherein the second chimeric polypeptide comprises a sequence that is 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 that is at least 90% identical to SEQ ID NO:308.

Embodiment G231. The multichain chimeric polypeptide of embodiment G230, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 that is 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 multichain chimeric polypeptide of any one of embodiments G1 and G234-G238, wherein the second chimeric polypeptide comprises a sequence that is 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 that is 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 that is 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 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 comprises the soluble tissue factor domain and the first of the paired affinity domains. A multi-chain chimeric polypeptide according to any one of embodiments G1-G133, which is positioned between a domain of

Embodiment G245. The first chimeric polypeptide 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 at least one of the one or more additional antigen binding domains. The multichain chimeric polypeptide of embodiment G244, further comprising a linker sequence between one and the first of the pair of affinity domains.

Embodiment G246. The multiple according to any one of embodiments G1-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. chain chimeric polypeptides.

Embodiment G247. According to embodiment G246, wherein at least one of the one or more additional target binding domains is directly adjacent to the first of the pair of affinity domains within the first chimeric polypeptide multi-chain chimeric polypeptide of.

Embodiment G248. The multiple 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 of the pair of affinity domains. chain chimeric polypeptides.

Embodiment G249. The multichain chimera of embodiment G246, wherein at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain within the first chimeric polypeptide Polypeptide.

Embodiment G250. The multichain 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. At least one of the one or more additional target binding domains is located at the N-terminus and/or C-terminus of the first chimeric polypeptide, wherein the one or more additional target binding domains are The multichain chimeric polypeptide of embodiment G246, at least one of which is positioned between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide.

Embodiment G252. At least one additional target binding domain of the one or more additional target binding domains located at the N-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment G251, which is directly contiguous to one domain.

Embodiment G253. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment G251, further comprising a sequence.

Embodiment G254. At least one additional target binding domain of the one or more additional target binding domains located at the C-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The multichain chimeric polypeptide of embodiment G251, which is directly contiguous to one domain.

Embodiment G255. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment G251, further comprising a sequence.

Embodiment G256. at least one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains is the soluble tissue factor domain and/or the affinity pair The multi-chain chimeric polypeptide of embodiment G251, wherein the domains are directly adjacent to the first domain.

Embodiment G257. the first chimeric polypeptide comprises: (i) the soluble tissue factor domain and one or more additional target binding domains positioned between the soluble tissue factor domain and the first of the pair of affinity domains; and/or (ii) between the first domain of the pair of affinity domains and the soluble tissue factor domain and the first domain of the pair of affinity domains The multichain chimeric polypeptide of embodiment G251, further comprising a linker sequence interposed with at least one of the additional target binding domains of embodiment G251.

Embodiment G258. Embodiments G44-G46, G57-G59, G76-G78, G96-G98, G109-, wherein the second chimeric polypeptide further comprises an additional target binding domain at the N-terminus or C-terminus of the second chimeric polypeptide The multichain chimeric polypeptide of any one of G111, G122-G124, and G131-G133.

Embodiment G259. The multichain chimeric polypeptide of embodiment G258, wherein the additional target binding domain is directly adjacent to a second of the pair of affinity domains within the second chimeric polypeptide.

Embodiment G260. The multichain of embodiment G258, wherein the second chimeric polypeptide further comprises a linker sequence between an additional target binding domain in the second chimeric polypeptide and the second of the pair of affinity domains. chimeric polypeptide.

Embodiment G261. The multichain chimeric polypeptide of embodiment G258, wherein the additional target binding domain is directly adjacent to the second target binding domain within the second chimeric polypeptide.

Embodiment G262. The multichain chimeric polypeptide of embodiment G258, wherein the second chimeric polypeptide further comprises a linker sequence between the additional target binding domain and the second target binding domain within the second chimeric polypeptide.

Embodiment G263. A composition comprising any of the multi-chain chimeric polypeptides of embodiments G1-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 of the multi-chain chimeric polypeptides of any one of embodiments G1-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 the nucleic acid of embodiment G323 or the vector of embodiment G267 or G268.

Embodiment G270. A method of producing a multi-chain chimeric polypeptide comprising:
culturing the cells of embodiment G269 in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide;
recovering the multi-chain chimeric polypeptide from the cells and/or the culture medium.

Embodiment G271. A multi-chain chimeric polypeptide produced by the method of embodiment G270.

Embodiment G272. The multichain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:97.

Embodiment G273. The multichain chimeric polypeptide of embodiment G272, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:97.

Embodiment G274. The multichain chimeric polypeptide of embodiment G273, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:97.

Embodiment G275. The multichain chimeric polypeptide of embodiment G274, wherein the soluble human tissue factor domain comprises a sequence that is 100% identical to SEQ ID NO:97.

Embodiment G276. The multichain chimeric polypeptide of embodiment G8, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:98.

Embodiment G277. The multichain chimeric polypeptide of embodiment G276, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:98.

Embodiment G278. The multichain chimeric polypeptide of embodiment G277, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:98.

Embodiment G279. The multichain 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 age-related disease or condition in a subject in need thereof, comprising administering to a subject identified as having an age-related disease or condition a therapeutically effective amount of one or more administering a natural killer (NK) cell activator of

Embodiment H2. A method of killing senescent cells 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 activating A method comprising administering a substance.

Embodiment H3. Senescent cells are senescent cancer cells, senescent monocytes, senescent lymphocytes, senescent astrocytes, senescent microglia, senescent neurons, senescent tissue fibroblasts, senescent skin fibroblasts, senescent keratinocytes, or other differentiated tissue-specific The method of embodiment H2, wherein the cell is a 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 as having an age-related disease or condition.

Embodiment H6. Embodiments H1 or H5, wherein the age-related disease or condition is selected from the group consisting of cancer, autoimmune disease, metabolic disease, neurodegenerative disease, cardiovascular disease, skin disease, progeria, and frailty disease. The method described in .

Embodiment H7. Cancer is solid tumor, hematologic tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome ( MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer cancer, non-small cell lung cancer, squamous cell head and neck cancer, 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 mellitus.

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 artery hypertension.

Embodiment H12. The method of embodiment H6, wherein the skin disorder is selected from the group consisting of wound healing, alopecia, wrinkles, senile lentigines, skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

Embodiment H13. The method of embodiment H6, wherein the progeria is selected from the group consisting of progeria and Hutchinson-Guilford progeria syndrome.

Embodiment H14. The method of embodiment H6, wherein the fragility disease is selected from the group consisting of fragility, response to vaccination, osteoporosis, and sarcopenia.

Embodiment H15. Age-related disease or condition is age-related macular degeneration, osteoarthritis, fat atrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, renal graft failure, liver fibrosis, bone loss, sarcopenia, age-related lung The method of embodiment H1 or H5, which is selected from the group of loss of tissue elasticity, osteoporosis, age-related renal dysfunction, and chemically induced renal dysfunction.

Embodiment H16. The method of embodiment H1 or H5, wherein the age-related disease or condition is diabetes mellitus type 2 or atherosclerosis.

Embodiment H17. The method of any one of embodiments H1-H16, wherein administering results in a decrease in the number of senescent cells in the target tissue in the subject.

Embodiment H18. The target tissue is selected from the group consisting of adipose tissue, pancreatic tissue, liver tissue, lung tissue, vascular system, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue. The method of embodiment H17.

Embodiment H19. The method of any one of embodiments H1-H18, wherein administration results in increased expression levels of CD25, CD69, MTOR-C1, SREBP1, IFN-γ, and granzyme B on activated NK cells.

Embodiment H20. A method of treating an age-related disease or condition in a subject in need of treatment of an age-related disease or condition, comprising administering to a subject identified as having an age-related disease or condition a therapeutically effective number of activated administering NK cells.

Embodiment H21. A method of killing senescent cells or reducing the number of senescent cells in a subject in need of killing senescent cells or reducing the number of senescent cells, comprising administering to the subject a therapeutically effective number of activated NK cells A method comprising:

Embodiment H22. Senescent cells are senescent cancer cells, senescent monocytes, senescent lymphocytes, senescent astrocytes, senescent microglia, senescent neurons, senescent tissue fibroblasts, senescent skin fibroblasts, senescent keratinocytes, or other differentiated tissue-specific The method of embodiment H21, wherein the cell is a dividing functional cell.

Embodiment H23. 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 as having an age-related disease or condition.

Embodiment H25. Embodiments H20 or H24, wherein the age-related disease or condition is selected from the group consisting of cancer, autoimmune disease, metabolic disease, neurodegenerative disease, cardiovascular disease, skin disease, progeria, and frailty disease. The method described in .

Embodiment H26. Cancer is solid tumor, hematologic tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome ( MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. .

Embodiment H27. 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 mellitus.

Embodiment H29. 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 artery hypertension.

Embodiment H31. The method of embodiment H25, wherein the skin disorder is selected from the group consisting of wound healing, alopecia, wrinkles, senile lentigines, skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

Embodiment H32. The method of embodiment H25, wherein the progeria is selected from the group consisting of progeria and Hutchinson-Guilford progeria syndrome.

Embodiment H33. The method of embodiment H25, wherein the fragility disease is selected from the group consisting of fragility, response to vaccination, osteoporosis, and sarcopenia.

Embodiment H34. Age-related disease or condition is age-related macular degeneration, osteoarthritis, fat atrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, renal graft failure, liver fibrosis, bone loss, sarcopenia, age-related lung The method of embodiment H20 or H24, which is selected from the group consisting of loss of tissue elasticity, osteoporosis, age-related renal dysfunction, and chemically-induced renal dysfunction.

Embodiment H35.
obtaining resting NK cells;
Contacting resting NK cells in vitro in a liquid culture medium containing one or more NK cell activators, which contact results in the generation of activated NK cells that are then administered to the subject. , contacting. The method of any one of embodiments H20-H34.

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 induced NK cells.

Embodiment H39. The method of embodiment H35, wherein the resting NK cells are haploidentical resting NK cells.

Embodiment H40. The method of any one of embodiments H35-H39, wherein the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

Embodiment H41. The method of any one of embodiments H35-H40, further comprising isolating the activated NK cells before the activated NK cells are administered to the subject.

Embodiment H42. 1. A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time comprising administering to the subject a therapeutically effective amount of one A method comprising administering the above natural killer (NK) cell activator.

Embodiment H43. 1. A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective number of activated administering the NK cells.

Embodiment H44.
obtaining resting NK cells;
Contacting resting NK cells in vitro in a liquid culture medium containing one or more NK cell activators, which contact results in the generation of activated NK cells that are then administered to the subject. , contacting. The method of embodiment H43.

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 induced NK cells.

Embodiment H48. The method of embodiment H44, wherein the resting NK cells are haploidentical resting NK cells.

Embodiment H49. The method of any one of embodiments H44-H48, wherein the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

Embodiment H50. The method of any one of embodiments H44-H49, further comprising isolating the activated NK cells before the activated NK cells are administered to the subject.

Embodiment H51. The method of any one of embodiments H42-H50, which provides an improvement in the subject's skin texture and/or appearance over said time period.

Embodiment H52. The method of embodiment H51, which results in a decrease in the rate of wrinkling of the subject's skin over said time period.

Embodiment H53. The method of embodiment H51 or H52, which results in improvement of the subject's skin pigmentation over said time period.

Embodiment H54. The method of any one of embodiments H51-H53, which results in an improvement in the subject's skin texture over said time period.

Embodiment H55. The method of any one of embodiments H42-H50, which provides an improvement in the texture and/or appearance of the subject's hair over said period of time.

Embodiment H56. The method of embodiment H55, which results in a decrease in the rate of gray hair formation in the subject over said time period.

Embodiment H57. The method of embodiment H55 or H56, which results in a decrease in the number of gray hairs in the subject over said time period.

Embodiment H58. The method of any one of embodiments H55-H57, which results in a decrease in the subject's rate of hair loss over time.

Embodiment H59. The method of any one of embodiments H55-H58, which results in improved texture of the subject's hair over said time period.

Embodiment H60. The method of any one of embodiments H42-H59, wherein said period of time is from about 1 month to about 10 years.

Embodiment H61. The method of any one of embodiments H42-H60, which results in a decrease in the number of senescent dermal fibroblasts in the subject's skin over said time period.

Embodiment H62. A method of helping treat obesity in a subject in need of help treating obesity over a period of time comprising administering to the subject a therapeutically effective amount of one or more natural killer (NK) cell activators. including, method.

Embodiment H63. A method of helping treat obesity in a subject in need of help treating obesity over a period of time, the method comprising administering to the subject a therapeutically effective number of activated NK cells.

Embodiment H64.
obtaining resting NK cells;
Contacting resting NK cells in vitro in a liquid culture medium containing one or more NK cell activators, which contact results in the generation of activated NK cells that are then administered to the subject. , contacting. The method of embodiment H63.

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 induced NK cells.

Embodiment H68. The method of embodiment H64, wherein the resting NK cells are haploidentical resting NK cells.

Embodiment H69. The method of any one of embodiments H64-H68, wherein the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

Embodiment H70. The method of any one of embodiments H64-H69, further comprising isolating the activated NK cells before the activated NK cells are administered to the subject.

Embodiment H71. The method of any one of embodiments H62-H70, which results in the subject losing weight over said time period.

Embodiment H72. The method of any one of embodiments H62-H71, which results in a reduction in the subject's body mass index (BMI) over said time period.

Embodiment H73. The method of any one of embodiments H62-H70, which results in a decrease in the rate of progression from prediabetes to type 2 diabetes in the subject.

Embodiment H74. The method of any one of embodiments H62-H70, which results in decreased fasting blood glucose levels in the subject.

Embodiment H75. The method of any one of embodiments H62-H70, which results in increased insulin sensitivity in the subject.

Embodiment H76. The method of any one of embodiments H62-H70, which results in a reduction in the severity of atherosclerosis in the subject.

Embodiment H77. The method of any one of embodiments H62-H76, wherein said period of time is from about 2 weeks to about 10 years.

Embodiment H78. At least one of the one or more NK cell activator(s) is IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor, IL-33 receptor, CD16, CD69, CD25, CD36, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4 , KIR2DS4, KIR2DS5, and KIR3DS1, the method of any one of embodiments H1-H19, H35-H42, H44-H62, and H64-H77.

Embodiment H79. Embodiment H78, wherein at least one of the one or more NK cell activators that result in IL-2 receptor activation is soluble IL-2 or an agonistic antibody that specifically binds IL-2 receptor. The method described in .

Embodiment H80. Embodiment H78, wherein at least one of the one or more NK cell activators that result in IL-7 receptor activation is soluble IL-7 or an agonistic antibody that specifically binds IL-7 receptor. The method described in .

Embodiment H81. Embodiment H78, wherein at least one of the one or more NK cell activators that result in IL-12 receptor activation is soluble IL-12 or an agonistic antibody that specifically binds IL-12 receptor. The method described in .

Embodiment H82. Embodiment H78, wherein at least one of the one or more NK cell activators that result in IL-15 receptor activation is soluble IL-15 or an agonist antibody that specifically binds IL-15 receptor. The method described in .

Embodiment H83. Embodiment H78, wherein at least one of the one or more NK cell activators that result in IL-21 receptor activation is soluble IL-21 or an agonist antibody that specifically binds IL-21 receptor. The method described in .

Embodiment H84. Embodiment H78, wherein at least one of the one or more NK cell activators that result in IL-33 receptor activation is soluble IL-33 or an agonistic antibody that specifically binds IL-33 receptor. The method described in .

Embodiment H85. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in CD16 receptor activation is an agonistic antibody that specifically binds CD16.

Embodiment H86. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in CD69 receptor activation is an agonistic antibody that specifically binds CD69.

Embodiment H87. According to embodiment H78, wherein at least one of the one or more NK cell activators that result in CD25, CD36, CD59 receptor activation is an agonistic antibody that specifically binds CD25, CD6, CD59. Method.

Embodiment H88. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in CD352 receptor activation is an agonistic antibody that specifically binds CD352.

Embodiment H89. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the NKp80 receptor is an agonistic antibody that specifically binds NKp80.

Embodiment H90. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the DNAM-1 receptor is an agonistic antibody that specifically binds DNAM-1.

Embodiment H91. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the 2B4 receptor is an agonistic antibody that specifically binds 2B4.

Embodiment H92. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the NKp30 receptor is an agonistic antibody that specifically binds NKp30.

Embodiment H93. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the NKp44 receptor is an agonistic antibody that specifically binds NKp44.

Embodiment H94. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the NKp46 receptor is an agonistic antibody that specifically binds NKp46.

Embodiment H95. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in NKG2D receptor activation is an agonistic antibody that specifically binds NKG2D.

Embodiment H96. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the KIR2DS1 receptor is an agonistic antibody that specifically binds KIR2DS1.

Embodiment H97. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the KIR2DS2/3 receptor is an agonistic antibody that specifically binds KIR2DS2/3.

Embodiment H98. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the KIR2DL4 receptor is an agonistic antibody that specifically binds KIR2DL4.

Embodiment H99. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the KIR2DS4 receptor is an agonistic antibody that specifically binds KIR2DS4.

Embodiment H100. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the KIR2DS5 receptor is an agonistic antibody that specifically binds KIR2DS5.

Embodiment H101. The method of embodiment H78, wherein at least one of the one or more NK cell activators that result in activation of the KIR3DS1 receptor is an agonistic antibody that specifically binds KIR3DS1.

Embodiment H102. At least one of the one or more NK cell activators is PD-1, TGF-beta receptor, TIGIT, CD1, TIM-3, Siglec-7, IRP60, Tactile, IL1R8, NKG2A/ Embodiments H1-H19, H35-H42, H44-, which result in decreased activation of one or more of KLRD1, KIR2DL1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2 H62, and the method of any one of H64-H101.

Embodiment H103. at least one of the one or more NK cell activators that results in decreased activation of PD-1, an antagonist antibody that specifically binds to PD-1, soluble PD-1, soluble PD-L1, or PD - The method of embodiment H102, which is an antibody that specifically binds to L1.

Embodiment H104. at least one of the one or more NK cell activators that results in decreased TGF-β receptor activation is a soluble TGF-β receptor, an antibody that specifically binds TGF-β, or TGF-β The method of embodiment H102, which is an antagonist antibody that specifically binds to the receptor.

Embodiment H105. at least one of the one or more NK cell activators that result in decreased activation of TIGIT is an antagonist antibody that specifically binds TIGIT, a soluble TIGIT, or an antibody that specifically binds a ligand of TIGIT , embodiment H102.

Embodiment H106. at least one of the one or more NK cell activators that result in decreased activation of CD1 is an antagonist antibody that specifically binds CD1, soluble CD1, or an antibody that specifically binds a ligand of CD1 , embodiment H102.

Embodiment H107. at least one of the one or more NK cell activators that results in decreased activation of TIM-3 is an antagonist antibody that specifically binds TIM-3, soluble TIM-3, or a ligand of TIM-3; The method of embodiment H102, which is a specifically binding antibody.

Embodiment H108. At least one of the one or more NK cell activators that results in decreased activation of Siglec-7 is an antagonist antibody that specifically binds to Siglec-7 or an antibody that specifically binds to a ligand of Siglec-7 The method of embodiment H102, wherein

Embodiment H109. Embodiment H102, wherein at least one of the one or more NK cell activators that result in decreased activation of IRP60 is an antagonist antibody that specifically binds IRP60 or an antibody that specifically binds a ligand of IRP60. The method described in .

Embodiment H110. Embodiment H102 wherein at least one of the one or more NK cell activators that result in decreased activation of Tactile is an antagonist antibody that specifically binds Tactile or an antibody that specifically binds a ligand of Tactile. The method described in .

Embodiment H111. Embodiment H102, wherein at least one of the one or more NK cell activators that result in decreased activation of IL1R8 is an antagonist antibody that specifically binds to IL1R8 or an antibody that specifically binds to a ligand of IL1R8. The method described in .

Embodiment H112. at least one of the one or more NK cell activators that results in decreased activation of NKG2A/KLRD1 is an antagonist antibody that specifically binds to NKG2A/KLRD1 or an antibody that specifically binds to a ligand of NKG2A/KLRD1 The method of embodiment H102, wherein

Embodiment H113. Embodiment H102, wherein at least one of the one or more NK cell activators that result in decreased activation of KIR2DL1 is an antagonist antibody that specifically binds KIR2DL1 or an antibody that specifically binds a ligand of KIR2DL1. The method described in .

Embodiment H114. at least one of the one or more NK cell activators that results in decreased activation of KIR2DL2/3 is an antagonist antibody that specifically binds to KIR2DL2/3 or an antibody that specifically binds to a ligand of KIR2DL2/3 The method of embodiment H102, wherein

Embodiment H115. Embodiment H102, wherein at least one of the one or more NK cell activators that result in decreased activation of KIR2DL5 is an antagonist antibody that specifically binds KIR2DL5 or an antibody that specifically binds a ligand of KIR2DL5. The method described in .

Embodiment H116. Embodiment H102, wherein at least one of the one or more NK cell activators that result in decreased activation of KIR3DL1 is an antagonist antibody that specifically binds KIR3DL1 or an antibody that specifically binds a ligand of KIR3DL1. The method described in .

Embodiment H117. Embodiment H102, wherein at least one of the one or more NK cell activators that result in decreased activation of KIR3DL2 is an antagonist antibody that specifically binds KIR3DL2 or an antibody that specifically binds a ligand of KIR3DL2. The method described in .

Embodiment H118. at least one of the one or more NK cell activators that results in decreased activation of ILT2/LIR-1 is an antagonist antibody that specifically binds to ILT2/LIR-1 or a ligand for ILT2/LIR-1; The method of embodiment H102, which is a specifically binding antibody.

Embodiment H119. At least one of the one or more NK cell activators that results in decreased activation of LAG-2 is an antagonist antibody that specifically binds LAG-2 or an antibody that specifically binds a ligand of LAG-2 The method of embodiment H102, wherein

Embodiment H120. At least one of the one or more NK cell activators is
(i) a first target binding domain;
(ii) a soluble tissue factor domain; and (iii) a second target binding domain.

Embodiment H121. The method 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-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-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-H128, wherein the first target binding domain and the second target binding domain specifically bind 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-H128, wherein the first target binding domain and the second target binding domain specifically bind different antigens.

Embodiment H133. The method of any one of embodiments H120-H132, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

Embodiment H134. The method of embodiment H133, wherein the first target binding domain and the second target binding domain are each antigen binding domains.

Embodiment H135. The method of embodiment H134, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment H136. one or both of the first target binding domain and the second target binding domain are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL -6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA , P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA The method of any one of embodiments H120-H135, wherein the method binds a target selected from the group consisting of receptors, MICB receptors, ULP16 binding protein receptors, CD155 receptors, and CD122 receptors.

Embodiment H137. The method of any one of embodiments H120-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. Soluble interleukin or cytokine proteins are 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-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. Embodiments wherein the interleukin or cytokine receptor is soluble TGF-beta receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL-4 receptor, or soluble IL-10 receptor The method described in H139.

Embodiment H141. The method of any one of embodiments H120-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 that is 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 that is 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 that is at least 95% identical to SEQ ID NO:93.

Embodiment H145. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method according to any one of H141 to H144.

Embodiment H146. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method described in H145.

Embodiment H147. The method of any one of embodiments H120-H146, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment H148. The method of any one of embodiments H120-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-H148, wherein the single-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

Embodiment H150. The method of any one of embodiments H120-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 are directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain.

Embodiment H153. 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; The method of embodiment H152.

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. Described in Embodiment H154, wherein one of the one or more additional target binding domains is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. the method of.

Embodiment H156. 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; The method of embodiment H154.

Embodiment H157. The method of embodiment H150, wherein the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus and C-terminus.

Embodiment H158. According to embodiment H157, wherein one of the one or more additional antigen binding domains at the N-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Method.

Embodiment H159. the single-chain chimeric polypeptide has 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; The method of embodiment H157, further comprising

Embodiment H160. According to embodiment H157, wherein one of the one or more additional antigen binding domains at the C-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. Method.

Embodiment H161. the single-chain chimeric polypeptide has 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; The method of embodiment H157, further comprising

Embodiment H162. any one of embodiments H150-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 the method described in Section 1.

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 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 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-H161, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind different antigens.

Embodiment H169. The method of any one of embodiments H150-H168, 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 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. one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD- 1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-beta receptor II (TGF -βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, specifically binds to a target selected from the group consisting of stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor; The method of any one of forms H150-H171.

Embodiment H173. any one of embodiments H150-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 the method described in Section 1.

Embodiment H174. Soluble interleukin or cytokine proteins are 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. Any of embodiments H150-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 The method described in 1.

Embodiment H176. Described in embodiment H175, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL-4 receptor, or soluble IL-10 receptor the method of.

Embodiment H177. At least one of the one or more NK cell activators comprises a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises
(c) a first chimeric polypeptide,
(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,
a second chimeric polypeptide comprising (i) a second of the pair of affinity domains, and (ii) a second target binding domain;
the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of a pair of affinity domains;
The method of any one of embodiments H1-H19, H35-H42, H44-H62, and H64-H77.

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 within 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 within the first chimeric polypeptide.

Embodiment H180. The method of any one of embodiments H177-H179, wherein the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide.

Embodiment H181. Any one of embodiments H177-H179, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide. the method described in Section 1.

Embodiment H182. The method of any one of embodiments H177-H181, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide.

Embodiment H183. Any of embodiments H177-H181, wherein the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide or the method of claim 1.

Embodiment H184. The method of any one of embodiments H177-H183, wherein the first target binding domain and the second target binding domain specifically bind 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-H183, wherein the first target binding domain and the second target binding domain specifically bind different antigens.

Embodiment H188. The method of any one of embodiments H177-H187, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

Embodiment H189. The method of embodiment H188, wherein the first target binding domain and the second target binding domain are each antigen binding domains.

Embodiment H190. The method of embodiment H188 or H189, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment H191. one or both of the first target binding domain and the second target binding domain are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL -6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA , P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA The method of any one of embodiments H177-H190, wherein the method specifically binds to a target selected from the group consisting of receptors, MICB receptors, ULP16 binding protein receptors, CD155 receptors, and CD122 receptors. .

Embodiment H192. 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 protein.

Embodiment H193. Soluble interleukin or cytokine proteins are 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. Described in embodiment H194, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL-4 receptor, or soluble IL-10 receptor the method of.

Embodiment H196. 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 comprises the soluble tissue factor domain and the first of the paired affinity domains. The method of any one of embodiments H177-H195, wherein the method is positioned between a domain of

Embodiment H197. The first chimeric polypeptide 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 at least one of the one or more additional antigen binding domains. The method of embodiment H196, further comprising a linker sequence between one and the first of the pair of affinity domains.

Embodiment H198. The method of any one of embodiments H177-H195, 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 H199. According to embodiment H198, wherein at least one of the one or more additional target binding domains is directly adjacent to the first of the pair of affinity domains in the first chimeric polypeptide the method of.

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 of the pair of affinity domains. .

Embodiment H201. The method of embodiment H198, wherein at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain within the first chimeric polypeptide.

Embodiment H202. 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 H203. At least one of the one or more additional target binding domains is positioned at the N-terminus and/or C-terminus of the first chimeric polypeptide, wherein the one or more additional target binding domains are The method of embodiment H198, wherein at least one of is positioned between the soluble tissue factor domain and the first of the pair of affinity domains in the first chimeric polypeptide.

Embodiment H204. At least one additional target binding domain of the one or more additional target binding domains located at the N-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The method of embodiment H203, which is directly adjacent to one domain.

Embodiment H205. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The method of embodiment H203, further comprising a sequence.

Embodiment H206. At least one additional target binding domain of the one or more additional target binding domains located at the C-terminus is the first target binding domain in the first chimeric polypeptide or the first of the pair of affinity domains The method of embodiment H203, which is directly adjacent to one domain.

Embodiment H207. a linker wherein the first chimeric polypeptide is positioned between at least one additional target binding domain within the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains The method of embodiment H203, further comprising a sequence.

Embodiment H208. at least one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains is the soluble tissue factor domain and/or the affinity pair The method of embodiment H203, wherein the domain is directly adjacent to the first domain.

Embodiment H209. the first chimeric polypeptide comprises: (i) the soluble tissue factor domain and one or more additional target binding domains positioned between the soluble tissue factor domain and the first of the pair of affinity domains; and/or (ii) between the first domain of the pair of affinity domains and the soluble tissue factor domain and the first domain of the pair of affinity domains The method of embodiment H203, further comprising a linker sequence interposed between and at least one of the additional target binding domains of.

Embodiment H210. The method of any one of embodiments H177-H209, 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 H211. Described in embodiment H210, wherein at least one of the one or more additional target binding domains is directly adjacent to a second domain of the pair of affinity domains within the second chimeric polypeptide the method of.

Embodiment H212. 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 of the pair of affinity domains , embodiment H210.

Embodiment H213. The method of embodiment H210, wherein at least one of the one or more additional target binding domains is directly adjacent to the second target binding domain within the second chimeric polypeptide.

Embodiment H214. 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 within the second chimeric polypeptide and the second target binding domain. The method described in .

Embodiment H215. any one of embodiments H196-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 the method described in Section 1.

Embodiment H216. 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 H217. 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 H218. 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 the same antigen.

Embodiment H219. 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 the same epitope.

Embodiment H220. 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 H221. The method of any one of embodiments H196-H214, wherein the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind different antigens.

Embodiment H222. The method of any one of embodiments H196-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 H223. 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 an antigen binding domain.

Embodiment H224. The method of embodiment H223, wherein the antigen binding domain comprises an scFv.

Embodiment H225. one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD- 1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-beta receptor II (TGF -βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like Embodiment H196 specifically binds to a target selected from the group consisting of the tyrosine kinase 3 ligand (FLT3L) receptor, the MICA receptor, the MICB receptor, the ULP16 binding protein receptor, the CD155 receptor, and the CD122 receptor. -H224.

Embodiment H226. 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 the method described in Section 1.

Embodiment H227. Soluble interleukin or cytokine proteins are 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 H228. Any 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 The method described in 1.

Embodiment H229. Described in embodiment H228, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL-4 receptor, or soluble IL-10 receptor the method of.

Embodiment H230. The method of any one of embodiments H196-H229, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

Embodiment H231. The method of embodiment H230, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93.

Embodiment H232. The method of embodiment H231, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO:93.

Embodiment H233. The method of embodiment H232, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO:93.

Embodiment H234. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method according to any one of H230-H233.

Embodiment H235. A soluble human tissue factor domain is
a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein;
an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein;
tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein;
an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein;
a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein;
An embodiment that does not include either arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. The method described in H234.

Embodiment H236. The method of any one of embodiments H196-H235, wherein the soluble tissue factor domain is incapable of binding Factor VIIa.

Embodiment H237. 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 H238. The method of any one of embodiments H196-H237, wherein the multichain chimeric polypeptide does not stimulate blood clotting in the mammal.

Embodiment H239. The method of any one of embodiments H196-H238, wherein the paired affinity domains are a sushi domain from the alpha chain of the human IL-15 receptor (IL-15Rα) and soluble IL-15.

Embodiment H240. The method of embodiment H239, wherein the soluble IL-15 has a D8N or D8A amino acid substitution.

Embodiment H241. The method of embodiment H239 or H240, wherein the human IL-15Rα is mature full-length IL-15Rα.

Embodiment H242. The paired affinity domains are selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. The method of any one of embodiments H196-H238, wherein the method is

Embodiment H243. At least one of the one or more NK cell activators comprises a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises
(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 of the pair of affinity domains, and (ii) a second target binding domain, comprising third and fourth chimeric polypeptides,
The first and second chimeric polypeptides and the third and fourth chimeric polypeptides associate via binding of the first and second domains of a pair of affinity domains, and two chimeric polypeptides associated through their Fc domains;
The method of any one of embodiments H1-H19, H35-H42, H44-H62, and H64-H77.

Embodiment H244. The method of embodiment H243, wherein the first target binding domain and the Fc domain are directly adjacent to each other within the first and second chimeric polypeptides.

Embodiment H245. 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 within the first and second chimeric polypeptides.

Embodiment H246. The method of any one of embodiments H243-H245, wherein the Fc domain and the first of the paired affinity domains are directly adjacent to each other within the first and second chimeric polypeptides.

Embodiment H247. Any of embodiments H243-H245, wherein the first chimeric polypeptide further comprises a linker sequence between the Fc domain and the first of the pair of affinity domains in the first and second chimeric polypeptides The method described in 1.

Embodiment H248. Any one of embodiments H243-H247, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the third and fourth chimeric polypeptides. Method.

Embodiment H249. wherein the third and fourth chimeric polypeptides further comprise a linker sequence between the second of the pair of affinity domains within the third and fourth chimeric polypeptides and the second target binding domain; The method of any one of forms H243-H247.

Embodiment H250. The method of any one of embodiments H243-H249, wherein the first target binding domain and the second target binding domain specifically bind 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 H253. The method of any one of embodiments H243-H249, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

Embodiment H254. The method of any one of embodiments H243-H253, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

Embodiment H255. The method of embodiment H254, wherein the first target binding domain and the second target binding domain are each antigen binding domains.

Embodiment H256. The method of embodiment H254 or H255, wherein the antigen binding domain comprises a scFv or single domain antibody.

Embodiment H257. one or both of the first target binding domain and the second target binding domain are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL -6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA , P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA The method of any one of embodiments H243-H256, wherein the method specifically binds to a target selected from the group consisting of receptors, MICB receptors, ULP16 binding protein receptors, CD155 receptors, and CD122 receptors. .

Embodiment H258. The method of any one of embodiments H243-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 H259. Soluble interleukin or cytokine proteins are 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 H260. The method of any one of embodiments H243-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 H261. Described in embodiment H260, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL-4 receptor, or soluble IL-10 receptor the method of.

Embodiment I1. A method of treating an age-related disease or condition in a subject in need thereof, comprising administering to a subject identified as having an age-related disease or condition a therapeutically effective amount of one or more administering a natural killer (NK) cell activator of

Embodiment I2. A method of killing senescent cells 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 activating A method comprising administering a substance.

Embodiment I3. The method of embodiment I1 or I2, wherein administering results in a decrease in the number of senescent cells in the target tissue in the subject.

Embodiment I4. The target tissue is selected from the group consisting of adipose tissue, pancreatic tissue, liver tissue, lung tissue, vascular system, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and secondary lymphoid organ tissue. The method of embodiment I3, wherein:

Embodiment I5. A method of treating an age-related disease or condition in a subject in need of treatment of an age-related disease or condition, comprising administering to a subject identified as having an age-related disease or condition a therapeutically effective number of activated administering NK cells.

Embodiment I6. A method of killing senescent cells or reducing the number of senescent cells in a subject in need of killing senescent cells or reducing the number of senescent cells, comprising administering to the subject a therapeutically effective number of activated NK cells A method comprising:

Embodiment I7. The method of any one of embodiments I1-I6, wherein the subject has been identified or diagnosed as having an age-related disease or condition.

Embodiment I8. According to embodiment I7, wherein the age-related disease or condition is selected from the group consisting of cancer, autoimmune disease, metabolic disease, neurodegenerative disease, cardiovascular disease, skin disease, progeria, and frailty disease. the method of.

Embodiment I9. Cancer is solid tumor, hematologic tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome ( MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer cancer, non-small cell lung cancer, squamous cell head and neck cancer, 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 mellitus.

Embodiment I12. The method of embodiment I8, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and dementia.

Embodiment I13. 9. The method of embodiment 8, wherein the cardiovascular disease is selected from the group consisting of coronary artery disease, atherosclerosis, and pulmonary artery hypertension.

Embodiment I14. The method of embodiment I8, wherein the skin disorder is selected from the group consisting of wound healing, alopecia, wrinkles, senile lentigines, skin thinning, xeroderma pigmentosum, and dyskeratosis congenita.

Embodiment I15. The method of embodiment I8, wherein the progeria is selected from the group consisting of progeria and Hutchinson-Guilford progeria syndrome.

Embodiment I16. The method of embodiment I8, wherein the fragility disease is selected from the group consisting of fragility, response to vaccination, osteoporosis, and sarcopenia.

Embodiment I17. Age-related disease or condition is osteoarthritis, fat atrophy, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, renal graft failure, liver fibrosis, bone loss, sarcopenia, age-related loss of lung tissue elasticity, osteoporosis The method of any one of embodiments I1-I6, wherein the method is selected from the group consisting of disease, age-related renal impairment, and chemically-induced renal impairment.

Embodiment I18. The method of any one of embodiments I1-I6, wherein the age-related disease or condition is diabetes mellitus type 2 or atherosclerosis.

Embodiment I19. 1. A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time comprising administering to the subject a therapeutically effective amount of one A method comprising administering the above natural killer (NK) cell activator.

Embodiment I20. 1. A method of improving skin and/or hair texture and/or appearance in a subject in need thereof over a period of time, comprising administering to the subject a therapeutically effective number of activated administering the NK cells.

Embodiment I21. The method of embodiment I19 or I20, which provides an improvement in the subject's skin texture and/or appearance over said period of time.

Embodiment I22. The method of embodiment I21, which results in a decrease in the rate of wrinkling of the subject's skin over said period of time.

Embodiment I23. The method of embodiment I21 or I22, which results in an improvement in the subject's skin pigmentation over said period of time.

Embodiment I24. The method of any one of embodiments I21-I23, which results in an improvement in the subject's skin texture over said period of time.

Embodiment I25. The method of any one of embodiments I20-I24, which provides an improvement in the texture and/or appearance of the subject's hair over said period of time.

Embodiment I26. The method of embodiment 125, which results in a decrease in the rate of gray hair formation in the subject over said time period.

Embodiment I27. The method of embodiment I25 or I26, which results in a decrease in the number of gray hairs in the subject over said time period.

Embodiment I28. The method of any one of embodiments I25-I27, which results in a decrease in the subject's rate of hair loss over time.

Embodiment I29. The method of any one of embodiments I25-I28, which results in an improvement in the subject's hair texture over said period of time.

Embodiment I30. The method of any one of embodiments I19-I29, which results in a decrease in the number of senescent dermal fibroblasts in the subject's skin over said period of time.

Embodiment I31. A method of helping treat obesity in a subject in need of help treating obesity over a period of time comprising administering to the subject a therapeutically effective amount of one or more natural killer (NK) cell activators. including, method.

Embodiment I32. A method of helping treat obesity in a subject in need of help treating obesity over a period of time, the method comprising administering to the subject a therapeutically effective number of activated NK cells.

Embodiment I33.
obtaining resting NK cells;
Contacting resting NK cells in vitro in a liquid culture medium containing one or more NK cell activators, which contact results in the generation of activated NK cells that are then administered to the subject. , contacting. The method of any one of embodiments I1-I32.

Embodiment I34. The method of embodiment I33, wherein the resting NK cells are genetically engineered NK cells with chimeric antigen receptors or recombinant T cell receptors.

Embodiment I35. The method of embodiment I33, further comprising introducing nucleic acid encoding the chimeric antigen receptor or recombinant T cell receptor into resting or activated NK cells prior to administration to the subject.

Embodiment I36. The method of any one of embodiments I31-I35, which results in the subject losing weight over said time period.

Embodiment I37. The method of any one of embodiments I31-I36, which results in a decrease in the subject's body mass index (BMI) over said time period.

Embodiment I38. The method of any one of embodiments I31-I35, which results in a decrease in the rate of progression from prediabetes to type 2 diabetes in the subject.

Embodiment I39. The method of any one of embodiments I31-I35, which results in decreased fasting blood glucose levels in the subject.

Embodiment I40. The method of any one of embodiments I31-I35, which results in increased insulin sensitivity in the subject.

Embodiment I41. The method of any one of embodiments I31-I35, which results in a reduction in the severity of atherosclerosis in the subject.

Embodiment I42.The one or more NK cell activators is IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, IL-21 receptor , IL-33 receptor, CD16, CD69, CD25, CD36, CD59, CD352, NKp80, DNAM-1, 2B4, NKp30, NKp44, NKp46, NKG2D, KIR2DS1, KIR2Ds2/3, KIR2DL4, KIR2DS4, KIR2DS5, and KIR3DS1 The method of any one of embodiments I1-I41, which results in activation of one or more of:

Embodiment I43. At least one of the one or more NK cell activators is PD-1, TGF-beta receptor, TIGIT, CD1, TIM-3, Siglec-7, IRP60, Tactile, IL1R8, NKG2A/ according to any one of embodiments I1-I42, which results in decreased activation of one or more of KLRD1, KIR2DL1, KIR2DL2/3, KIR2DL5, KIR3DL1, KIR3DL2, ILT2/LIR-1, and LAG-2 the method of.

Embodiment I44. At least one of the one or more NK cell activators is
(i) a first target binding domain;
(ii) a soluble tissue factor domain; and (iii) a second target binding domain.

Embodiment I45. The method 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-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-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. At least one of the one or more NK cell activators comprises a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises
(a) a first chimeric polypeptide,
(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,
a second chimeric polypeptide comprising (i) a second of the pair of affinity domains, and (ii) a second target binding domain;
the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of a pair of affinity domains;
The method according to any one of I1-I41.

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 within 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 within the first chimeric polypeptide.

Embodiment I52. The method of any one of embodiments I49-I51, wherein the soluble tissue factor domain and the first of the paired affinity domains are directly adjacent to each other within the first chimeric polypeptide.

Embodiment I53. Any one of embodiments I49-I51, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first of the pair of affinity domains within the first chimeric polypeptide the method described in Section 1.

Embodiment I54. The method of any one of embodiments I49-I53, wherein the second domain and the second target binding domain of the pair of affinity domains are directly adjacent to each other within the second chimeric polypeptide.

Embodiment I55. Any of embodiments I49-I53, wherein the second chimeric polypeptide further comprises a linker sequence between the second of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide or the method of claim 1.

Embodiment I56. At least one of the one or more NK cell activators comprises a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises
(a) first and second chimeric polypeptides, each comprising:
(i) a first target binding domain;
(ii) an Fc domain, and (iii) a first of a pair of affinity domains, first and second chimeric polypeptides,
(b) third and fourth chimeric polypeptides, each comprising:
(i) a second of the pair of affinity domains, and (ii) a second target binding domain, comprising third and fourth chimeric polypeptides,
The first and second chimeric polypeptides and the third and fourth chimeric polypeptides associate via binding of the first and second domains of a pair of affinity domains, and two chimeric polypeptides associated through their Fc domains;
The method of any one of embodiments I1-I41.

Embodiment I57. one or both of the first target binding domain and the second target binding domain are CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB , IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM , BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand , DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor , MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor. the method of.

Embodiment I58. The method of any one of embodiments I44-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. Soluble interleukin or cytokine proteins are 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-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. Described in embodiment I60, wherein the soluble receptor is soluble TGF-beta receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL-4 receptor, or soluble IL-10 receptor the method of.

Embodiment I62. The method of any one of embodiments I44-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-I55, wherein the soluble tissue factor domain comprises or consists of a sequence derived from wild-type soluble human tissue factor.

Claims (194)

A method of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject, comprising treatment with one or more agents that result in decreased activation of TGF-beta receptors. The method, comprising administering an effective amount to the subject. A method of reducing spontaneous and/or treatment-induced accumulation of senescent cells in a subject, comprising administering to said subject a therapeutically effective amount of one or more agents that result in decreased activation of TGF-beta receptors. The method as described above, comprising: A method of reducing the levels of markers of spontaneous and/or treatment-induced senescence in a subject, comprising administering to said subject a therapeutically effective amount of one or more agents that result in decreased activation of TGF-β receptors. The above method, comprising administering. A method of reducing spontaneous and/or treatment-induced senescent cell activity in a subject, comprising administering to said subject a therapeutically effective amount of one or more agents that result in decreased activation of TGF-beta receptors. The method as described above, comprising: 1. 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 one or more resulting in decreased activation of TGF-beta receptors. administering to said subject a therapeutically effective amount of an agent of 6. The method of any one of claims 1-5, wherein the subject has been previously diagnosed or identified as having an age-related disease or an inflammatory disease. 7. The method of claim 6, wherein said age-related disease is inflammatory senescence-related. said age-related disease is Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, inflammatory bowel disease, disc degeneration, osteoarthritis, type 2 diabetes mellitus, fat atrophy, lipodystrophy, Atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, hypertrophy Age-related lung tissue elastic loss, age-related macular degeneration, cachexia, glomerulosclerosis, liver 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. The age-related disease is solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplasia syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, The cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. 6. The method according to 6. The inflammatory disease is 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, 7. The method of claim 6, selected from the group consisting of Guillain-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders, and cancer therapy-related cognitive impairment. The method of any one of claims 1-10, wherein said therapy-induced senescent cells are chemotherapy-induced senescent cells. Said administration of said one or more agents that result in decreased activation of TGF-β receptors results in a decrease in the number or activity of spontaneous and/or treatment-induced senescent cells in target tissues in said subject. , the method according to any one of claims 1 to 11. The target tissue is adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and secondary lymph tissue. 13. The method of claim 12, selected from the group consisting of organ tissue. 14. The method of any one of claims 1-13, wherein the TGFβ receptor is TGF-β receptor II (TGF-βRII). 14. The method of any one of claims 1-13, wherein the TGFβ receptor is TGF-βRIII. at least one of said one or more agents that result in decreased activation of a TGF-beta receptor is a soluble TGF-beta receptor, an extracellular domain of a TGF-beta receptor, TGF-beta specific 16. An antibody that binds, an antagonist antibody that binds a TGF-beta receptor, an agent that binds LAP, or an agent that binds the TGF-beta/LAP complex, according to any one of claims 1-15. the method of. the one or more agents that result in decreased TGF-β receptor activation decrease TGF-β receptor activation through binding to LAP or to the TGF-β/LAP complex; 17. The method of claim 16. at least one of said one or more agents that result in decreased activation of a TGF-beta receptor is a multi-chain chimeric polypeptide, said multi-chain chimeric polypeptide comprising:
(e) a first chimeric polypeptide,
(i) a first target binding domain;
(ii) a soluble tissue factor domain, and (iii) a first of a pair of affinity domains,
(f) a second chimeric polypeptide,
(i) a second of a pair of affinity domains; and (ii) a second target binding domain;
one or both of said first target binding domain and said second target binding domain specifically bind to a ligand of a TGF-β receptor, or said first target binding domain and said second target binding domain one or both of the target binding domains of is an antagonist antigen binding domain that specifically binds to the TGF-β receptor;
A method according to any one of claims 1-15. 19. The method of claim 18, wherein said TGF-beta receptor is TGF-betaRII. 19. The method of claim 18, wherein said TGF-beta receptor is TGF-beta RIII. 21. The method of any one of claims 18-20, wherein said first target binding domain and said soluble tissue factor domain are directly adjacent to each other within said first chimeric polypeptide. 21. Any one of claims 18-20, wherein said first chimeric polypeptide further comprises a linker sequence between said first target binding domain and said soluble tissue factor domain within said first chimeric polypeptide. The method described in section. 23. Any one of claims 18-22, wherein said soluble tissue factor domain and said first domain of said pair of affinity domains are directly adjacent to each other within said first chimeric polypeptide. Method. Claims 18-, wherein said first chimeric polypeptide further comprises a linker sequence between said soluble tissue factor domain within said first chimeric polypeptide and said first of said pair of affinity domains. 23. The method of any one of clauses 22. 25. Any one of claims 18-24, wherein said second domain of said pair of affinity domains and said second target binding domain are directly adjacent to each other within said second chimeric polypeptide. described method. 4. wherein said second chimeric polypeptide further comprises a linker sequence between said second of said pair of affinity domains and said second target binding domain within said second chimeric polypeptide. 25. The method of any one of 18-24. 27. The method of any one of claims 18-26, wherein said first target binding domain and said second target binding domain specifically bind to the same antigen. 27. The method of any one of claims 18-26, wherein said first target binding domain and said second target binding domain specifically bind to different antigens. 29. The method of any one of claims 18-28, wherein said first chimeric polypeptide further comprises one or more additional target binding domains. 30. The method of any one of claims 18-29, wherein said second chimeric polypeptide further comprises one or more additional target binding domains. The method of any one of claims 18-30, wherein said soluble tissue factor domain is a soluble human tissue factor domain. 32. The method of claim 31, wherein said soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93. The method according to any one of claims 18 to 32, wherein said pair of affinity domains is a sushi domain derived from the alpha chain of human IL-15 receptor (IL15Rα) and soluble IL-15. 34. The method of claim 33, wherein said soluble IL-15 has a D8N or D8A amino acid substitution. 35. The method of claim 33 or 34, wherein said soluble IL-15 comprises mutations to reduce or eliminate IL-15 activity. The paired affinity domains are from barnase and barnstar, PKA and AKAP, an adapter/docking tag module based on mutated RNase I fragments, and a SNARE module based on the interaction of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. The method of any one of claims 18-32, selected from the group consisting of: 18- wherein said first domain or said second domain of a pair of affinity domains is an antigen binding domain that specifically binds to a soluble common gamma chain family cytokine or a common gamma chain family cytokine receptor 33. The method of any one of clauses 32. 38. The method of any one of claims 18-37, wherein said first target binding domain and/or said second target binding domain comprises a soluble TGF-beta receptor. 39. The method of claim 38, wherein said soluble TGF-beta receptor is soluble TGF-beta RII. said soluble TGF-βRII 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; are separated by a linker. 41. The method of claim 40, wherein said soluble TGF-βRII 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. 42. The method of claim 41, wherein said soluble TGF-βRII comprises a first sequence of SEQ ID NO:183 and a second sequence of SEQ ID NO:183. 41. The method of claim 40, wherein the linker comprises the sequence of SEQ ID NO:102. 40. The method of claim 39, wherein said soluble TGF-βRII comprises a sequence that is at least 80% identical to SEQ ID NO:188. 45. The method of claim 44, wherein said soluble TGF-βRII comprises a sequence that is at least 90% identical to SEQ ID NO:188. 46. The method of claim 45, wherein said soluble TGF-βRII comprises the sequence of SEQ ID NO:188. 19. The method of claim 18, wherein said first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:236. 48. The method of claim 47, wherein said first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:236. 49. The method of claim 48, wherein said first chimeric polypeptide comprises the sequence of SEQ ID NO:236. 19. The method of claim 18, wherein said second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:193. 51. The method of claim 50, wherein said first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:236. 52. The method of claim 51, wherein said second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:193. 53. The method of claim 52, wherein said second chimeric polypeptide comprises the sequence of SEQ ID NO:193. 54. The method of claim 53, wherein said first chimeric polypeptide comprises the sequence of SEQ ID NO:236. at least one of the one or more agents that results in decreased activation of the TGF-β receptor;
(i) a first target binding domain;
(ii) a soluble tissue factor domain, and (iii) a second target binding domain, a single chain chimeric polypeptide,
one or both of said first target binding domain and said second target binding domain specifically bind to a ligand of a TGF-β receptor, or said first target binding domain and said second target binding domain one or both of the target binding domains of is an antagonist antigen binding domain that specifically binds to the TGF-β receptor;
A method according to any one of claims 1-15. 56. The method of claim 55, wherein said TGF-beta receptor is TGF-betaRII. 56. The method of claim 55, wherein said TGF-beta receptor is TGF-beta RIII. 58. The method of any one of claims 55-57, wherein said first target binding domain and said soluble tissue factor domain are directly adjacent to each other. 58. The method of any one of claims 55-57, wherein said single chain chimeric polypeptide further comprises a linker sequence between said first target binding domain and said soluble tissue factor domain. 60. The method of any one of claims 55-59, wherein said soluble tissue factor domain and said second target binding domain are directly adjacent to each other. 60. The method of any one of claims 55-59, wherein said single chain chimeric polypeptide further comprises a linker sequence between said soluble tissue factor domain and said second target binding domain. 62. The method of any one of claims 55-61, wherein said first target binding domain and said second target binding domain specifically bind to the same antigen. 62. The method of any one of claims 55-61, wherein said first target binding domain and said second target binding domain specifically bind to different antigens. The method of any one of claims 55-63, wherein said soluble tissue factor domain is a soluble human tissue factor domain. 65. The method of claim 64, wherein said soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93. 66. The method of any one of claims 55-65, wherein said single chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and/or C-terminus. 67. The method of any one of claims 55-66, wherein said first target binding domain and/or said second target binding domain comprises a soluble TGF-beta receptor. 68. The method of claim 67, wherein said soluble TGF-beta receptor is soluble TGF-beta RII. said soluble TGF-βRII 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; are separated by a linker. 70. The method of claim 69, wherein said soluble TGF-βRII 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. 71. The method of claim 70, wherein said soluble TGF-βRII comprises a first sequence of SEQ ID NO:183 and a second sequence of SEQ ID NO:183. 70. The method of claim 69, wherein said linker comprises the sequence of SEQ ID NO:102. 73. The method of claim 72, wherein said soluble TGF-βRII comprises a sequence that is at least 80% identical to SEQ ID NO:188. 74. The method of claim 73, wherein said soluble TGF-βRII comprises a sequence that is at least 90% identical to SEQ ID NO:188. 75. The method of 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, comprising administering to the subject two or more doses of the one or more agents that result in decreased activation of a TGF-beta receptor. 77. The method of claim 76, wherein any two consecutive doses of said two or more doses are administered about one week to about one year apart. 78. The method of claim 77, wherein any two consecutive doses of said two or more doses are administered about one week to about six months apart. 79. The method of claim 78, wherein any two consecutive doses of said two or more doses are administered about one week to about two months apart. 80. The method of claim 79, wherein any two consecutive doses of said two or more doses are administered about one week to about one month apart. 81. The method of any one of claims 76-80, wherein said two or more doses are administered by subcutaneous administration. 81. The method of any one of claims 76-80, wherein said two or more doses are administered by intramuscular administration. 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 said two or more doses are administered over a period of about 1 year to about 50 years. 85. The method of claim 84, wherein said two or more doses are administered over a period of about 1 year to about 40 years. 86. The method of claim 85, wherein said two or more doses are administered over a period of about 1 year to about 30 years. 87. The method of claim 86, wherein said two or more doses are administered over a period of about 1 year to about 20 years. 88. The method of claim 87, wherein said two or more doses are administered over a period of about 1 year to about 10 years. 89. Any one of claims 1-88, wherein the first administration of said one or more agents that result in decreased TGF-beta receptor activation begins when said subject reaches at least 30 years of age. Method. 90. The method of claim 89, wherein the first administration of said one or more agents that result in decreased TGF-beta receptor activation begins when said subject reaches at least 40 years of age. 91. The method of claim 90, wherein the first administration of said one or more agents that result in decreased TGF-beta receptor activation begins when said subject reaches at least 50 years of age. 92. The method of claim 91, wherein the first administration of said one or more agents that result in decreased TGF-beta receptor activation begins when said subject reaches at least 60 years of age. Each of said two or more doses ranges from about 0.01 mg/kg of each agent that results in decreased activation of TGF-β receptors to about 10 mg/kg of each agent that results in decreased activation of TGF-β receptors. 93. The method of any one of claims 1-92, administered in a dosage of kg. Each of said two or more doses ranges from about 0.02 mg/kg of each agent that results in decreased activation of TGF-β receptors to about 5 mg/kg of each agent that results in decreased activation of TGF-β receptors. 94. The method of claim 93, administered in a dosage of kg. 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 age-related disease or an inflammatory disease. The method of any one of claims 1-3 and 9-95, wherein said subject has not been previously treated with a chemotherapeutic agent. 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. A method of killing or reducing the number of naturally occurring and/or treatment-induced senescent cells in a subject, comprising: The above method, comprising administering to a subject. 1. A method of reducing spontaneous and/or treatment-induced accumulation of senescent cells in a subject, comprising administering to said subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. the aforementioned method. 1. A method of reducing the levels of markers of spontaneous and/or treatment-induced senescence in a subject, comprising administering to said subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. The method above. 1. A method of reducing naturally occurring and/or treatment-induced senescent cell activity in a subject, comprising administering to said subject a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators. the aforementioned method. 1. A method of reducing the level or activity of naturally occurring and/or treatment-induced senescent cell-derived SASP factors in a subject, comprising administering a therapeutically effective amount of one or more common gamma chain family cytokine receptor activators to said subject. The above method, comprising administering to 103. The method of any one of claims 98-102, wherein the subject has been previously diagnosed or identified as having an age-related disease or an inflammatory disease. 104. The method of claim 103, wherein said age-related disease is inflammatory senescence-related. said age-related disease is Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, inflammatory bowel disease, disc degeneration, osteoarthritis, type 2 diabetes mellitus, fat atrophy, lipodystrophy, Atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, hypertrophy Age-related lung tissue elastic loss, age-related macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis , neurodegeneration, stroke, blood-brain barrier disorders, cancer, dementia, vascular disease, susceptibility to infection, chronic inflammation, and renal dysfunction. The age-related disease is solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplasia syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, The cancer selected from the group consisting of ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. 103. The method according to 103. The inflammatory disease is 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, 104. The method of claim 103, wherein the method is selected from the group consisting of Guillain-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders, and cancer therapy-related cognitive impairment. 108. The method of any one of claims 98-107, wherein said therapy-induced senescent cells are chemotherapy-induced senescent cells. Claims 98-108, wherein administration of said one or more common gamma chain family cytokine receptor activators results in a reduction in the number of spontaneous and/or treatment-induced senescent cells in target tissues in said subject. The method according to any one of . The target tissue is adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vasculature, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and secondary lymph tissue. 110. The method of claim 109, selected from the group consisting of organ tissue. at least one of the one or more common gamma chain family cytokine receptor activators is combined with a complex of a common gamma chain family cytokine or functional fragment thereof and the common gamma chain family cytokine or functional fragment thereof; 111. The method of any one of claims 98-110, which is a conjugate with a specifically binding antibody or antibody fragment. at least one of the one or more common gamma chain family cytokine receptor activators
(i) a first target binding domain;
(ii) a soluble tissue factor domain, and (iii) a second target binding domain, a single chain chimeric polypeptide,
one or both of said first target binding domain and said second target binding domain is a soluble common gamma chain family cytokine, an agonist antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common an antigen binding domain that specifically binds a gamma chain family cytokine receptor or a common gamma chain family cytokine;
The method of any one of claims 98-110. 113. The method of claim 112, wherein one or both of said first target binding domain and said second target binding domain comprise a soluble common gamma chain family cytokine. Claim 113, wherein said 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. The method described in . 115. The method of claim 114, wherein one or both of said first target binding domain and said second target binding domain comprise an agonist antigen binding domain that specifically binds to a common gamma chain family cytokine receptor. . 116. The claim 115, wherein said 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. described method. 117. The method of claim 115 or 116, wherein said agonist antigen binding domain is scFv, VHH, or VNAR. 118. The method of any one of claims 112-117, wherein said first target binding domain and said soluble tissue factor domain are directly adjacent to each other. 118. The method of any one of claims 112-117, wherein said single chain chimeric polypeptide further comprises a linker sequence between said first target binding domain and said soluble tissue factor domain. The method of any one of claims 112-119, wherein said soluble tissue factor domain and said second target binding domain are directly adjacent to each other. The method of any one of claims 112-119, wherein said single chain chimeric polypeptide further comprises a linker sequence between said soluble tissue factor domain and said second target binding domain. The method of any one of claims 112-121, wherein said first target binding domain and said second target binding domain specifically bind to the same antigen. 123. The method of claim 122, wherein said first target binding domain and said second target binding domain specifically bind to the same epitope. 124. The method of claim 123, wherein said first target binding domain and said second target binding domain comprise the same amino acid sequence. The method of any one of claims 112-121, wherein said first target binding domain and said second target binding domain specifically bind to different antigens. The method of any one of claims 112-125, wherein said soluble tissue factor domain is a soluble human tissue factor domain. 127. The method of claim 126, wherein said soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93. A method according to any one of claims 112 to 127, wherein said single chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and/or C-terminus. at least one of said one or more common gamma chain family cytokine receptor activators is a multi-chain chimeric polypeptide, said multi-chain chimeric polypeptide comprising:
(a) a first chimeric polypeptide,
(i) a first target binding domain;
(ii) a soluble tissue factor domain, and (iii) a first of a pair of affinity domains,
(b) a second chimeric polypeptide,
(i) a second of a pair of affinity domains; and (ii) a second target binding domain;
one or both of said first target binding domain and said second target binding domain is a soluble common gamma chain family cytokine, an agonist antigen binding domain that specifically binds to a common gamma chain family cytokine receptor, a soluble common an antigen binding domain that specifically binds a gamma chain family cytokine receptor or a common gamma chain family cytokine;
The method of any one of claims 98-110. 130. The method of claim 129, wherein one or both of said first target binding domain and said second target binding domain comprise a soluble common gamma chain family cytokine. Claim 130, wherein said 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. The method described in . 130. The method of claim 129, wherein one or both of said first target binding domain and said second target binding domain comprise an agonist antigen binding domain that specifically binds to a common gamma chain family cytokine receptor. . 133. according to claim 132, wherein said 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 described method. 134. The method of claim 132 or 133, wherein said agonist antigen binding domain is scFv, VHH, or VNAR. 135. The method of any one of claims 129-134, wherein said first target binding domain and said soluble tissue factor domain are directly adjacent to each other within said first chimeric polypeptide. 135. Any one of claims 129-134, wherein said first chimeric polypeptide further comprises a linker sequence between said first target binding domain and said soluble tissue factor domain within said first chimeric polypeptide. The method described in section. 137. Any one of claims 129-136, wherein said soluble tissue factor domain and said first domain of said pair of affinity domains are directly adjacent to each other within said first chimeric polypeptide. Method. claims 129-, wherein said first chimeric polypeptide further comprises a linker sequence between said soluble tissue factor domain and said first of said pair of affinity domains within said first chimeric polypeptide 136. The method according to any one of clauses 136 to 136. 139. Any one of claims 129-138, wherein said second domain of said pair of affinity domains and said second target binding domain are directly adjacent to each other within said second chimeric polypeptide. described method. 129. The second chimeric polypeptide further comprises a linker sequence between said second of said pair of affinity domains and said second target binding domain within said second chimeric polypeptide. 138. The method of any one of claims 1-138. 141. The method of any one of claims 129-140, wherein said first target binding domain and said second target binding domain specifically bind to the same antigen. 141. The method of any one of claims 129-140, wherein said first target binding domain and said second target binding domain specifically bind to different antigens. The method of any one of claims 129-142, wherein said first chimeric polypeptide further comprises one or more additional target binding domains. 144. The method of any one of claims 129-143, wherein said second chimeric polypeptide further comprises one or more additional target binding domains. The method of any one of claims 129-144, wherein said soluble tissue factor domain is a soluble human tissue factor domain. 146. The method of claim 145, wherein said soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:93. 147. The method of any one of claims 129-146, wherein said pair of affinity domains is a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. said pair of affinity domains from the group consisting of barnase and barnstar, PKA and AKAP, adapter/docking tag modules based on mutated RNase I fragments, and SNARE modules based on interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. 147. The method of any one of claims 129-146, selected. Claim 129- wherein said first domain or said second domain of a pair of affinity domains is an antigen binding domain that specifically binds to a soluble common gamma chain family cytokine or a common gamma chain family cytokine receptor 146. The method of any one of clauses 146 to 146. 111. The method of any one of claims 98-110, wherein at least one of said one or more common gamma chain family cytokine receptor activators is soluble IL-15, or an IL-15 agonist. 151. The method of claim 150, wherein said soluble IL-15 is at least 90% identical to SEQ ID NO:82. 151. The method of claim 150, wherein said 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 said portion of said soluble IL-15R is a portion of IL-15Rα. 154. The method of claim 153, wherein said portion of said soluble IL-15Rα is the sushi domain of IL-15Rα. The method of any one of claims 152-154, wherein said IL-15 agonist further comprises an Fc domain. 151. The method of claim 150, wherein said IL-15 agonist comprises a fusion protein comprising IL-15 and a sushi domain from IL-15Rα. 111. The method of any one of claims 98-110, wherein one of said one or more common gamma chain family cytokine receptor activators is a soluble IL-2 or IL-2 agonist. 111. Any one of claims 98-110, wherein one of said one or more common gamma chain family cytokine receptor activators is an antibody or antigen-binding antibody fragment that specifically binds to a common gamma chain family cytokine. The method described in section. 159. The method of any one of claims 98-158, comprising administering one, two or more doses of said one or more common gamma chain family cytokine receptor activators to said subject. 160. The method of claim 159, wherein any two consecutive doses of said two or more doses are administered about one week to about one year apart. 161. The method of claim 160, wherein any two consecutive doses of said two or more doses are administered about one week to about six months apart. 162. The method of claim 161, wherein any two consecutive doses of said two or more doses are administered about one week to about two months apart. 163. The method of claim 162, wherein any two consecutive doses of said two or more doses are administered about one week to about one month apart. 164. The method of any one of claims 159-163, wherein said one, two or more doses are administered by subcutaneous administration. 164. The method of any one of claims 159-163, wherein said two or more doses are administered by intramuscular administration. 166. The method of any one of claims 159-165, wherein said two or more doses are administered over a period of about 1 year to about 60 years. 167. The method of claim 166, wherein said two or more doses are administered over a period of about 1 year to about 50 years. 168. The method of claim 167, wherein said two or more doses are administered over a period of about 1 year to about 40 years. 169. The method of claim 168, wherein said two or more doses are administered over a period of about 1 year to about 30 years. 169. The method of claim 169, wherein said two or more doses are administered over a period of about 1 year to about 20 years. 171. The method of claim 170, wherein said two or more doses are administered over a period of about 1 year to about 10 years. Each of the two or more doses is administered at a dosage of about 0.01 mg/kg of each common gamma chain family cytokine receptor activator to about 10 mg/kg of each common gamma chain family cytokine receptor activator. , the method of any one of claims 98-171. Each of the two or more doses is administered at a dosage of about 0.02 mg/kg of each common gamma chain family cytokine receptor activator to about 5 mg/kg of each common gamma chain family cytokine receptor activator. 173. The method of claim 172. 174. The method of any one of claims 98-173, wherein the first administration of the one or more common gamma chain family cytokine receptor activators begins when the subject reaches at least 30 years of age. 175. The method of claim 174, wherein said first administration of said one or more common gamma chain family cytokine receptor activators begins when said subject reaches at least 40 years of age. 176. The method of claim 175, wherein said first administration of said one or more common gamma chain family cytokine receptor activators begins when said subject reaches at least 50 years of age. 177. The method of claim 176, wherein said first administration of said one or more common gamma chain family cytokine receptor activators begins when said subject reaches at least 60 years of age. 178. The method of any one of claims 98-102 and 108-177, wherein said subject has not been diagnosed or identified as having an age-related disease or an inflammatory disease. The method of any one of claims 98-102 and 108-178, wherein said subject has not been previously treated with a chemotherapeutic agent. The method of any one of claims 98-102 and 108-178, wherein said subject has not been previously treated with a therapeutic agent that induces cellular senescence. 181. The method of any one of claims 98-180, further comprising administering to said subject at least one or more agents that result in decreased activation of TGF-beta receptors. the one or more agents that result in decreased activation of the TGF-beta receptor is a soluble TGF-beta receptor, an extracellular domain of a TGF-beta receptor, an antibody that specifically binds to TGF-beta, TGF - an antagonist antibody that binds to a beta receptor, an agent that binds to LAP, or an agent that binds to the TGF-beta/LAP complex. wherein said one or more agents that result in decreased TGF-β activation decrease TGF-β receptor activation through binding to LAP or to the TGF-β/LAP complex. 182. 147. The method of any one of claims 31, 32, 64, 65, 126, 127, 145, and 146, wherein said soluble human tissue factor domain does not initiate blood clotting. 184. Any one of claims 1-184, further comprising administering an additional therapeutic agent selected from the group of combinations of agents such as checkpoint inhibitors, chemotherapeutic agents, and therapeutic antibodies. Method. The method of any one of claims 55-75, wherein said single-chain chimeric polypeptide is stable in human serum at 37°C for at least 10 days. 55. The method of any one of claims 18-54, wherein said multichain chimeric polypeptide is stable in human serum at 37°C for at least 10 days. The method of any one of claims 112-128, wherein said single-chain chimeric polypeptide does not have significant clotting activity. The method of any one of claims 129-149, wherein said multichain chimeric polypeptide does not have significant clotting activity. 189. The method of any one of claims 1-189, which results in rejuvenation of aged immune cells in said subject. 191. The method of claim 190, wherein said rejuvenation of said aged immune cells results in a reduction in the number of diseased cells or infectious agents in said subject. The aged immune cells are aged NK cells, aged NKT cells, aged T cells, aged B cells, aged monocytes, aged macrophages, aged neutrophils, aged basophils, aged 192. The method of claim 190 or 191, comprising one or more of eosinophils, senescent Kupffer cells, and senescent microgial cells. 192. The method of claim 191, wherein said diseased cells comprise cancer cells, virus-infected cells, and intracellular bacteria-infected cells. 192. The method of claim 191, wherein said infectious agents include viruses, bacteria, fungi, and parasites.

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