RU2792671C2 - Poly-specific binding proteins targeted to caix, ano1, mesothelin, trop2, cea, and claudine-18.2 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to the field of biotechnology, in particular to poly-specific antibodies; it can be used in medicine. The invention discloses new proteins, which specifically bind to NKG2D receptor, CD16, and CEA tumor-specific agent.

EFFECT: invention can be used in medical practice as a therapeutic agent in the treatment of a row of malignant neoplasms, for example gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer.

24 cl, 37 dwg, 14 tbl, 10 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit and priority of U.S. Application No. 62/464,341, filed February 27, 2017, U.S. Application No. 62/464,344, filed February 27, 2017, U.S. Application No. 62/464,347, filed February 27 2017, U.S. Application No. 62/467,557 filed March 6, 2017, U.S. Application No. 62/473,652 filed March 20, 2017, and U.S. Application No. 62/473,659 filed March 20, 2017, in full the contents of each of which are hereby incorporated by reference in their entirety for all purposes.

SEQUENCE LIST

[0002] This application contains a sequence listing, which is presented in electronic form in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on February 27, 2018, is named DFY-012WO_ST25.txt and is 212,480 bytes in size.

FIELD OF TECHNOLOGY

[0003] The invention relates to polyspecific binding proteins that bind to the NKG2D receptor, CD16, and to a tumor-specific antigen selected from carbonic anhydrase 9 (CAIX), anoctamine-1 (ANO1), mesothelin, trophoblast cell surface antigen (TROP2), carcinoembryonic antigen (CEA) and claudin-18.2.

State of the art

[0004] Malignancy continues to be a serious health problem, despite significant research efforts and scientific advances for the treatment of this disease, which are reported in the literature. Some of the most commonly diagnosed types of cancer include prostate cancer, breast cancer, and lung cancer. Prostate cancer is the most common form of malignant neoplasm in men. Breast cancer remains the leading cause of death in women. Small cell lung cancer remains one of the deadliest malignant neoplasms. Existing treatments for these types of cancers are not effective in all patients and/or may have significant side effects. Other types of malignancies also remain difficult to treat using existing therapies.

[0005] Antitumor immunotherapy is desirable because it is highly specific and can promote the destruction of tumor cells using the patient's own immune system. Fusion proteins, such as T cell recruiting bispecific proteins, are antitumor immunotherapeutic agents described in the literature that bind to tumor cells and T cells to facilitate destruction of tumor cells. Antibodies that bind to certain tumor-specific antigens and to certain immune cells have been described in the literature. See, for example, WO 2016/134371 and WO 2015/095412.

[0006] Natural killer (NK) cells are a component of the innate immune system and make up approximately 15% of circulating lymphocytes. NK cells penetrate virtually all tissues and were originally characterized by their ability to effectively kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells by means similar to cytotoxic T cells, ie, by cytolytic granules that contain perforin and granzymes, and by cell death receptor pathways. Activated NK cells also secrete inflammatory cytokines such as IFN-γ and chemokines that promote the recruitment of other white blood cells to the target tissue.

[0007] NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autocells, their activity is inhibited by activation of the killer cell's immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign cells or tumor cells, they are activated through their activation receptors (eg, NKG2D, NCR, DNAM1). NK cells are also activated by the constant region of some immunoglobulins via CD16 receptors on their surface. The overall sensitivity of NK cells to activation depends on the sum of stimulatory and inhibitory signals.

[0008] Carbonic anhydrase 9 (CAIX) is a zinc metalloenzyme and belongs to a family of enzymes that are involved in the reversible hydration of carbon dioxide to form bicarbonate and hydrogen ions. CAIX has limited expression in normal tissues, but is overexpressed in a wide range of solid tumors and plays an important role in tumor development. CAIX, induced by the hypoxic tumor microenvironment, functions to combat the detrimental effects of high levels of glycolytic metabolism in tumor cells by maintaining an intracellular pH favorable for tumor cell growth and survival, while at the same time creating an acidic extracellular space that facilitates tumor cell invasiveness. So far, overexpression of CAIX has been found in renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, as well as in common epithelial malignancies such as carcinomas of the esophagus, lung, and colon. intestines, kidneys, cervix, and non-small cell lung carcinoma.

[0009] Mesothelin is a glycosylphosphatidylinositol (GPI) attached to a cell surface protein and is expressed in mesothelial cells of the pleura, peritoneum, and pericardium. In addition to its normal expression, mesothelin is overexpressed in a variety of cancers including mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, cholangiocarcinoma, gastric cancer, uterine serous carcinoma, thymus carcinoma, and acute myeloid leukemia. It is hypothesized that mesothelin may play an important role in tumor development, including cell adhesion, cell survival/proliferation, and chemoresistance.

[0010] Anoctamine-1 (ANO1) is a voltage-sensitive calcium-activated chloride channel. It is also known by other names such as TMEM16, TAOS2, ORAOV2 and DOG-1. Calcium-activated chloride channels function in many physiological processes, including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction, and fertilization. ANO1 is potentially involved in epithelial fluid secretion, olfactory and phototransduction, neuronal and cardiac excitability, and regulation of vascular tone, including intestinal motility. ANO1 is also highly expressed in some cancers where it is thought to increase cell proliferation, cell migration, and metastasis, e.g., esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumor (GIST), head and neck squamous cell carcinoma (HNSCC). ), pancreatic cancer, breast cancer, prostate cancer and sarcoma. ~85% of HNSCC tumors are found to be ANO1-positive, and HNSCC patients with high levels of ANO1 tumor expression have reduced overall survival; it has also been found that ANO1 overexpression is closely associated with prostate cancer and poor prognosis in breast cancer.

[0011] TROP2 is a transmembrane glycoprotein and Ca2+ signal transducer. In addition to its expression in some normal tissues, TROP2 is overexpressed in a variety of cancers, including breast, lung, gastric, colorectal, pancreatic, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma, and ovarian carcinoma. It transmits signals to tumor cells about proliferation, invasion and survival.

[0012] Carcinoembryonic antigen (CEA), also known as CD66e or CEACAM5, is a member of the immunoglobulin superfamily. It is a large cell surface glycoprotein that primarily serves as a cell adhesion molecule that mediates intercellular contact. In addition to cell adhesion and migration functions, CEA is overexpressed in a high percentage of human malignancies, including 90% of gastrointestinal, colorectal, and pancreatic cancers, 70% of non-small cell lung cancers, and 50% of breast cancers. It has been shown that CEA overexpression correlates with oncogenicity and increased tumor invasiveness.

[0013] Claudin-18.2 is one of two isoforms of Claudin-18, a major component of tight junctions that regulate the permeability, barrier function, and polarity of epithelial layers. Claudin-18.2 is only expressed in short-lived differentiated gastric mucosal cells in healthy tissues. However, it is overexpressed in up to 80% of gastrointestinal adenocarcinomas and 60% of pancreatic tumors. In addition, its expression has been observed in selected groups of people with esophageal cancer, non-small cell lung carcinoma, ovarian cancer, colon cancer, and some forms of bile duct carcinoma.

SUMMARY OF THE INVENTION

[0014] The invention relates to polyspecific binding proteins that bind to CAIX, ANO1, mesothelin, TROP2, CEA or Claudin-18.2 on a tumor cell and to the NKG2D receptor and CD16 receptor on natural killer cells. Such proteins may interact with more than one type of NK-activating receptor and may block natural ligand binding to NKG2D. In some embodiments, the protein may be an NK cell agonist in humans and other species such as rodents and cynomolgus monkeys. Various aspects and embodiments of the invention are described in more detail below.

[0015] Accordingly, one aspect of the invention relates to a protein that includes a first antigen-binding site that binds to NKG2D; a second antigen-binding site that binds to CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2; and the Fc domain of an antibody, a CD16-binding portion thereof, or a third antigen-binding site that binds to CD16. Each antigen-binding site may include an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., located as in an antibody or fused together with scFv), or one or more antigen-binding sites may be a single domain antibody, such as a VHH antibody, such as a camel antibody , or a VNAR antibody similar to those found in cartilaginous fish.

[0016] In one embodiment, the first antigen-binding site that binds to NKG2D comprises the heavy chain variable domain linked to SEQ ID NO: 1, such as by at least 90% amino acid sequence (e.g., 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 1 and/or including amino acid sequences identical to those of CDR1 (SEQ ID NO: 51), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 53) SEQ ID NO: 1. The heavy chain variable domain associated with SEQ ID NO: 1 can be associated with various light chain variable domains. chains to form the NKG2D binding site. For example, the first antigen binding site that includes the heavy chain variable domain associated with SEQ ID NO: 1 may further include a light chain variable domain selected from any of the sequences associated with SEQ ID NO: 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 40. (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 1, and a light chain variable domain with amino acid sequences at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any of sequences selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 22, 24, 26, 28, 30, 32, 34, 36 and 40. Alternatively, the first antigen binding site may include the heavy chain variable domain linked to SEQ ID NO: 41 and the variab the light chain domain associated with SEQ ID NO: 42. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 41 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 54), CDR2 (SEQ ID NO: 55) and CDR3 (SEQ ID NO: 56) SEQ ID NO: 41. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 42 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 57), CDR2 (SEQ ID NO: 58) and CDR3 (SEQ ID NO: 59) SEQ ID NO: 42. In other embodiments, the first antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 43 and a light chain variable domain associated with SEQ ID NO: : 44. For example, the heavy chain variable domain of the first antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 43 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 60), CDR2 (SEQ ID NO: 61) and CDR3 (SEQ ID NO: 62) of SEQ ID NO: 43. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or 100%) is identical to SEQ ID NO: 44 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 63), CDR2 (SEQ ID NO: 64) and CDR3 (SEQ ID NO: 65) SEQ ID NO: 44. In other embodiments, the first antigen binding site may include a heavy chain variable domain linked to SEQ ID NO: 45 and a light chain variable domain linked to SEQ ID NO: 46. For example, the heavy chain variable domain of the first antigen binding site it may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 45 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 66), CDR2 (SEQ ID NO: 67) and CDR3 (SEQ ID NO: 68) of SEQ ID NO: 45. Similarly, light chain variable domain the second antigen-binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical SEQ ID NO: 46 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 69), CDR2 (SEQ ID NO: 70) and CDR3 (SEQ ID NO: 71) of SEQ ID NO: 46.

[0017] Alternatively, the first antigen-binding site may include a heavy chain variable domain associated with SEQ ID NO: 47 and a light chain variable domain associated with SEQ ID NO: 48, for example, by having amino acid sequences of at least 90 % (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 47 and SEQ ID NO: 48, respectively. In another embodiment, the first antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 49 and a light chain variable domain associated with SEQ ID NO: 50, for example, due to the presence of amino acid sequences of at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 49 and SEQ ID NO: 50, respectively .

[0018] In other embodiments, the first antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 132 and a light chain variable domain associated with SEQ ID NO: 133. For example, the heavy chain variable domain of the first antigen binding site may be , at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 132 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 134), CDR2 (SEQ ID NO: 135), and CDR3 (SEQ ID NO: 136) of SEQ ID NO: 132. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO : 133 and/or includes amino acid sequences identical to the CDR1 (SEQ ID NO: 137), CDR2 (SEQ ID NO: 138) and CDR3 (SEQ ID NO: 139) sequences of SEQ ID NO: 133.

[0019] In other embodiments, the first antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 140 and a light chain variable domain associated with SEQ ID NO: 141. For example, the heavy chain variable domain of the first antigen binding site may be , at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 140 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 142), CDR2 (SEQ ID NO: 143), and CDR3 (SEQ ID NO: 144) of SEQ ID NO: 140. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO : 141 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 145), CDR2 (SEQ ID NO: 146) and CDR3 (SEQ ID NO: 147) of SEQ ID NO: 141.

[0020] In other embodiments, the first antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 148 and a light chain variable domain associated with SEQ ID NO: 149. For example, the heavy chain variable domain of the first antigen binding site may be , at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 148 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 150), CDR2 (SEQ ID NO: 151), and CDR3 (SEQ ID NO: 152) of SEQ ID NO: 148. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO : 149 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 153), CDR2 (SEQ ID NO: 154) and CDR3 (SEQ ID NO: 155) of SEQ ID NO: 149.

[0021] In other embodiments, the first antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 156 and a light chain variable domain associated with SEQ ID NO: 157. For example, the heavy chain variable domain of the first antigen binding site may be , at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 156 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 158), CDR2 (SEQ ID NO: 159), and CDR3 (SEQ ID NO: 160) of SEQ ID NO: 156. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO : 157 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 161), CDR2 (SEQ ID NO: 162) and CDR3 (SEQ ID NO: 163) of SEQ ID NO: 157.

[0022] In other embodiments, the first antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 164 and a light chain variable domain associated with SEQ ID NO: 165. For example, the heavy chain variable domain of the first antigen binding site may be , at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 164 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 166), CDR2 (SEQ ID NO: 167), and CDR3 (SEQ ID NO: 168) of SEQ ID NO: 164. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO : 165 and/or includes amino acid sequences identical to those of CDR1 (SEQ ID NO: 169), CDR2 (SEQ ID NO: 170) and CDR3 (SEQ ID NO: 171) of SEQ ID NO: 165.

[0023] The second antigen binding site can bind to CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2. In some embodiments, the second antigen binding site binds to CAIX and includes a heavy chain variable domain and a light chain variable domain that can be associated to form a CAIX binding site. For example, the second antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 72 and a light chain variable domain associated with SEQ ID NO: 73. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 72 and/or include amino acids sequences identical to CDR1 (SEQ ID NO: 74), CDR2 (SEQ ID NO: 75), and CDR3 (SEQ ID NO: 76) of SEQ ID NO: 72. Similarly, the light chain variable domain of the second antigen binding site may be at least at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 73 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 77), CDR2 (SEQ ID NO: 78) and CDR3 (SEQ ID NO: 79) sequences of SEQ ID NO: 73.

[0024] Alternatively, the second antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 80 and a light chain variable domain associated with SEQ ID NO: 81. For example, the heavy chain variable domain of the second antigen binding site may be, at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 80 and/ or includes amino acid sequences identical to the CDR1 (SEQ ID NO: 82), CDR2 (SEQ ID NO: 83), and CDR3 (SEQ ID NO: 84) sequences of SEQ ID NO: 80. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 81 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 85), CDR2 (SEQ ID NO: 86) and CDR3 (SEQ ID NO: 87) of SEQ ID NO: 81.

[0025] Alternatively, the second antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 88 and a light chain variable domain associated with SEQ ID NO: 89. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 88 and/ or includes amino acid sequences identical to the CDR1 (SEQ ID NO: 90), CDR2 (SEQ ID NO: 91), and CDR3 (SEQ ID NO: 92) sequences of SEQ ID NO: 88. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 89 and/or includes amino acid sequences identical to the CDR1 (SEQ ID NO: 93), CDR2 (SEQ ID NO: 94) and CDR3 (SEQ ID NO: 95) sequences of SEQ ID NO: 89.

[0026] In some embodiments, the second antigen binding site binds to ANO1 and includes a heavy chain variable domain and a light chain variable domain that can bind to form an ANO1 binding site. For example, the second antigen binding site may include a complex of a heavy chain variable domain associated with SEQ ID NO: 97 and a light chain variable domain associated with SEQ ID NO: 98. For example, the heavy chain variable domain of the second antigen binding site may be at least , 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 97 and/or includes amino acid sequences identical to the CDR1 (SEQ ID NO: 99), CDR2 (SEQ ID NO: 100) and CDR3 (SEQ ID NO: 101) sequences of SEQ ID NO: 97. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 98 and /or includes amino acid sequences identical to the CDR1 (SEQ ID NO: 102), CDR2 (SEQ ID NO: 103) and CDR3 (SEQ ID NO: 104) sequences of SEQ ID NO: 98.

[0027] In some embodiments, the second antigen binding site binds to mesothelin and includes a heavy chain variable domain and a light chain variable domain, which can be associated to form a mesothelin binding site. For example, the second antigen binding site may include a set of a heavy chain variable domain associated with SEQ ID NO: 106 and a light chain variable domain associated with SEQ ID NO: 107. For example, the heavy chain variable domain of the second antigen binding site may be at least , 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 106 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 109), and CDR3 (SEQ ID NO: 110) of SEQ ID NO: 106. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 107 and/ or includes amino acid sequences identical to CDR1 (SEQ ID NO: 111), CDR2 (SEQ ID NO: 112), and CDR3 (SEQ ID NO: 113) of SEQ ID NO: 107. Alternatively, the second antigen-binding site may include a set op of the heavy chain variable domain associated with SEQ ID NO: 114 and the light chain variable domain associated with SEQ ID NO: 115. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 114 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 116), CDR2 (SEQ ID NO: 117), and CDR3 (SEQ ID NO: 118) of SEQ ID NO: 115. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% ( e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) is identical to SEQ ID NO: 115 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 119), CDR2 (SEQ ID NO: 120), and CDR3 (SEQ ID NO: 121) of SEQ ID NO: 115. Alternatively, the second antigen-binding site may include a set of heavy chain variable domain linked to SEQ ID N O: 122, and the light chain variable domain associated with SEQ ID NO: 123. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 122 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 124), CDR2 (SEQ ID NO: 124). NO: 125) and CDR3 (SEQ ID NO: 126) of SEQ ID NO: 122. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 123 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 127), CDR2 ( SEQ ID NO: 128) and CDR3 (SEQ ID NO: 129) of SEQ ID NO: 123.

[0028] In some embodiments, the second antigen binding site binds to TROP2 and includes a heavy chain variable domain and a light chain variable domain that can bind to form a TROP2 binding site. For example, the second antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 172 and a light chain variable domain associated with SEQ ID NO: 173. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 172 and/or includes amino acids sequences identical to CDR1 (SEQ ID NO: 174), CDR2 (SEQ ID NO: 175), and CDR3 (SEQ ID NO: 176) of SEQ ID NO: 172. Similarly, the light chain variable domain of the second antigen binding site may be at least at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 173 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 177), CDR2 (SEQ ID NO: 178), and CDR3 (SEQ ID NO: 179) of SEQ ID NO: 173. Alternatively, the second antigen-binding site may include variable up to the heavy chain variable domain associated with SEQ ID NO: 180 and the light chain variable domain associated with SEQ ID NO: 181. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 180 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO : 182), CDR2 (SEQ ID NO: 183), and CDR3 (SEQ ID NO: 184) of SEQ ID NO: 180. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 181 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 185), CDR2 (SEQ ID NO: 186), and CDR3 (SEQ ID NO: 187) of SEQ ID NO: 181. Alternatively, the second antigen binding site may include a heavy chain variable domain linked to SEQ ID NO: 188, and variable domain th chain associated with SEQ ID NO: 189. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, %, 96%, 97%, 98%, 99%, or 100%) is identical to SEQ ID NO: 188, and the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%), identical to SEQ ID NO: 189. Alternatively, the second antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 190, and the light chain variable domain associated with SEQ ID NO: 191. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 190 and the light chain variable domain of the second antigen binding site may be at least 90% (e.g. , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9 8%, 99%, or 100%), identical to SEQ ID NO: 191. Alternatively, the second antigen binding site may include a heavy chain variable domain linked to SEQ ID NO: 192 and a light chain variable domain linked to SEQ ID NO: 193 For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100%) is identical to SEQ ID NO: 192, and the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 193.

[0029] In some embodiments, the second antigen binding site binds to CEA and includes a heavy chain variable domain and a light chain variable domain that may be associated to form a CEA binding site. For example, the second antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 195 and a light chain variable domain associated with SEQ ID NO: 196. For example, the heavy chain variable domain of the second antigen binding site may be at least , 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 195 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 197), CDR2 (SEQ ID NO: 198), and CDR3 (SEQ ID NO: 199) of SEQ ID NO: 195. Similarly, the light chain variable domain of the second antigen binding site can be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 196 and/ or includes amino acid sequences identical to CDR1 (SEQ ID NO: 200), CDR2 (SEQ ID NO: 201), and CDR3 (SEQ ID NO: 202) of SEQ ID NO: 196. Alternatively, the second antigen-binding site may include variable d The heavy chain domain associated with SEQ ID NO: 203; and the light chain variable domain associated with SEQ ID NO: 204. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 203 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO : 205), CDR2 (SEQ ID NO: 206), and CDR3 (SEQ ID NO: 207) of SEQ ID NO: 203. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 204 and/or contain amino acid sequences identical to CDR1 (SEQ ID NO: 208), CDR2 (SEQ ID NO: 209) and CDR3 (SEQ ID NO: 210) of SEQ ID NO: 204. Alternatively, the second antigen binding site may include a heavy chain variable domain linked to SEQ ID NO: 211, and variable domain chain associated with SEQ ID NO: 212. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, , 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 211 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 213), CDR2 (SEQ ID NO: 214) and CDR3 ( SEQ ID NO: 215) of the sequence of SEQ ID NO: 212. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) is identical to SEQ ID NO: 196 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 216), CDR2 (SEQ ID NO: 217) and CDR3 (SEQ ID NO: 218) sequences of SEQ ID NO: 212.

[0030] In some embodiments, the second antigen binding site binds to Claudin-18.2 and includes a heavy chain variable domain and a light chain variable domain that can be associated to form a Claudin-18.2 binding site. For example, the second antigen binding site may include a heavy chain variable domain associated with SEQ ID NO: 220 and a light chain variable domain associated with SEQ ID NO: 221. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 220 and/or includes amino acids sequences identical to CDR1 (SEQ ID NO: 222), CDR2 (SEQ ID NO: 223), and CDR3 (SEQ ID NO: 224) of SEQ ID NO: 220. Similarly, the light chain variable domain of the second antigen binding site may be at least at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 221 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 225), CDR2 (SEQ ID NO: 226), and CDR3 (SEQ ID NO: 227) of SEQ ID NO: 221. For example, the second antigen binding site may include a variable domain heavy chain associated with SEQ ID NO: 228; and a light chain variable domain associated with SEQ ID NO: 229. For example, the heavy chain variable domain of the second antigen binding site may be at least 90% (e.g., 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 228 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 230), CDR2 (SEQ ID NO: 231), and CDR3 (SEQ ID NO: 232) of SEQ ID NO: 228. Similarly, the light chain variable domain of the second antigen binding site may be at least 90% (e.g., 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) is identical to SEQ ID NO: 229 and/or includes amino acid sequences identical to CDR1 (SEQ ID NO: 233), CDR2 (SEQ ID NO: 234) and CDR3 (SEQ ID NO: 235) of SEQ ID NO: 229.

[0031] In some embodiments, the second antigen binding site includes a light chain variable domain having an amino acid sequence identical to the amino acid sequence of the light chain variable domain present in the first antigen binding site.

[0032] In some embodiments, the protein comprises a portion of an antibody Fc domain sufficient to bind CD16, wherein the antibody Fc domain contains hinge and CH2 domains and/or amino acid sequences at least 90% identical to amino acid sequence 234-332 human IgG antibody sequences.

[0033] Compositions containing one of these proteins are provided; cells containing one or more nucleic acids expressing these proteins, and methods for enhancing tumor cell death using these proteins.

[0034] Another aspect of the invention relates to a method of treating cancer in a patient. The method includes administering to a patient in need thereof a therapeutically effective amount of a polyspecific binding protein as described herein. Cancers to be treated with CAIX-targeted polyspecific binding proteins include, for example, renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, tumors of the esophagus, lung, colon colon, kidney, cervical and non-small cell lung carcinoma. Cancers treatable with mesothelin-targeted polyspecific binding proteins include, for example, mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, cholangiocarcinoma, gastric cancer, uterine serous carcinoma, thymic carcinoma, and acute myeloid leukemia. . Cancers treatable with ANO1-targeted polyspecific binding proteins include, for example, esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumor (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, breast cancer , prostate cancer and sarcoma. Cancers to be treated with TROP2-targeted polyspecific binding proteins include, for example, breast, lung, stomach, colorectal, pancreatic, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma, and ovarian carcinoma. Cancers to be treated with CEA-targeted polyspecific binding proteins include, for example, gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. Cancers that can be treated using claudin-18.2-targeted polyspecific binding proteins include, for example, esophageal cancer, non-small cell lung carcinoma, ovarian cancer, colon cancer, and several forms of bile duct carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 represents a heterodimeric polyspecific antibody. Each arm can be either a NKG2D binding domain or one of the tumor specific antigen binding domains selected from CAIX, ANO1, mesothelin, TROP2, CEA and Claudin-18.2 binding domains. In some embodiments, the NKG2D- and tumor-specific antigen-binding domains may share a common light chain.

[0036] FIG. 2 represents a heterodimeric polyspecific antibody. Either NKG2D- or a tumor-specific antigen-binding domain selected from the CAIX, ANO1, mesothelin, TROP2, CEA and Claudin-18.2 binding domains can take the scFv format (right arm).

[0037] FIG. 3 is line graphs showing the binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D in an ELISA assay.

[0038] FIG. 4 is line graphs showing the binding affinity of NKG2D binding domains (listed as clones) to recombinant cynomolgus monkey NKG2D in an ELISA assay.

[0039] FIG. 5 is line graphs showing the binding affinity of NKG2D binding domains (listed as clones) to recombinant mouse NKG2D in an ELISA assay.

[0040] FIG. 6 are histograms showing, by flow cytometry, the binding of NKG2D binding domains (listed as clones) to EL4 cells expressing human NKG2D, showing a fold of mean fluorescence intensity (MFI) relative to background.

[0041] FIG. 7 are histograms showing, by flow cytometry, the binding of NKG2D binding domains (listed as clones) to EL4 cells expressing mouse NKG2D, showing a fold of mean fluorescence intensity (MFI) relative to background.

[0042] FIG. 8 is a line graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc by competition with the native ULBP-6 ligand.

[0043] FIG. 9 is a line graph showing the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant human NKG2D-Fc by competition with natural MICA ligand.

[0044] FIG. 10 are line graphs demonstrating the specific binding affinity of NKG2D binding domains (listed as clones) to recombinant mouse NKG2D-Fc by competition with the natural ligand Rae-1 delta.

[0045] FIG. 11 are histograms showing human NKG2D activation by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF alpha positive cells that express human NKG2D-CD3 zeta fusion proteins.

[0046] FIG. 12 are histograms showing mouse NKG2D activation by NKG2D binding domains (listed as clones) by quantifying the percentage of TNF alpha positive cells that express mouse NKG2D-CD3 zeta fusion proteins.

[0047] FIG. 13 are histograms showing human NK cell activation by NKG2D binding domains (listed as clones).

[0048] FIG. 14 are histograms showing human NK cell activation by NKG2D binding domains (listed as clones).

[0049] FIG. 15 are histograms showing the activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0050] FIG. 16 are histograms showing the activation of mouse NK cells by NKG2D binding domains (listed as clones).

[0051] FIG. 17 are histograms showing the cytotoxic effect of NKG2D binding domains (listed as clones) on tumor cells.

[0052] FIG. 18 are histograms showing the melting point of NKG2D binding domains (listed as clones) measured by differential scanning fluorometry.

[0053] FIG. 19A-19C are histograms of synergistic NK cell activation using CD16 and NKG2D binding. FIG. 19A shows the level of CD107a; FIG. 19B shows the level of IFN-γ; FIG 19C shows the level of production of CD107a and IFN-γ. Plots show mean (n=2) ± SD. Data represent five independent experiments using five different healthy donors.

[0054] FIG. 20 is TriNKET in the form of Triomab, which is a trifunctional bispecific antibody that maintains an IgG-like form. This chimera consists of two half antibodies, each with one light and one heavy chain, which are derived from two parental antibodies. The form of Triomab may be a heterodimeric construct containing 1/2 rat antibody and 1/2 mouse antibody.

[0055] FIG. 21 is a TriNKET in the form of a common light chain (LC) KiH, which includes a ridge-in-gap (KIH) technology. KiH is a heterodimer containing 2 target-binding Fabs 1 and 2 and an Fc stabilized by heterodimerization mutations. TriNKET in KiH format can be a heterodimeric construct with 2 Fabs binding to target 1 and target 2 and contains 2 different heavy chains and a common light chain that pairs with both heavy chains.

[0056] FIG. 22 is a TriNKET in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the domains that bind to the target of two monoclonal antibodies via flexible naturally occurring linkers to form a tetravalent IgG-like molecule. DVD-Ig™ is a homodimeric construct in which a variable domain targeting antigen 2 is fused to the N-terminus of a Fab variable domain targeting antigen 1. The construct contains a normal Fc.

[0057] FIG. 23 is a TriNKET in the form of an orthogonal Fab interface (Ortho-Fab), which is a heterodimeric construct that contains 2 target 1 and target 2 binding Fab fragments fused to an Fc. LC-HC pairing is provided by an orthogonal interface. Heterodimerization is provided by mutations in Fc.

[0058] FIG. 24 is TrinKET in 2-in-1 Ig format.

[0059] FIG. 25 is a TriNKET in ES form, which is a heterodimeric construct containing 2 different Fab fragments binding to target 1 and target 2 fused to Fc. Heterodimerization is mediated by electrostatic regulation mutations in Fc.

[0060] FIG. 26 is TriNKET in Fab exchange form: antibodies that exchange Fab fragments by replacing the heavy chain and the attached light chain (half-molecule) with a heavy-light chain pair from another molecule to produce bispecific antibodies. KiH is a heterodimer containing 2 target-binding Fabs 1 and 2 and an Fc stabilized by heterodimerization mutations.

[0061] FIG. 27 is a TriNKET in the form of a SEED Body, which is a heterodimer containing 2 target-binding Fab fragments 1 and 2 and FC stabilized by heterodimerization mutations.

[0062] FIG. 28 is a LuZ-Y TriNKET in which the leucine zipper domain is used to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing 2 different target-binding scFabs 1 and 2 fused to Fc. Heterodimerization is mediated by leucine zipper motifs fused to the C-terminus of Fc.

[0063] FIG. 29 is TriNKET in Cov-X-Body form.

[0064] FIG. 30A-30B are TriNKETs in ĸλ-Body forms which are heterodimeric constructs with two different Fab fused Fc stabilized heterodimerization mutations: Fab 1 targeting antigen 1 contains kappa LC and second Fab targeting antigen 2 contains lambda LC. FIG. 30A is an illustrative depiction of one form of ĸλ-Body; FIG. 30B is an illustrative representation of another ĸλ-Body.

[0065] FIG. 31 is a heterodimeric Oasc-Fab construct that includes a Fab that binds to target 1 and a scFab that binds to target 2 fused to Fc. Heterodimerization is provided by mutations in Fc.

[0066] FIG. 32 is a DuetMab which is a heterodimeric construct containing 2 different Antigen-binding Fabs 1 and 2 and an Fc stabilized by heterodimerization mutations. Fab 1 and 2 contain different SS-bridges that ensure proper light chain (LC) and heavy chain (HC) pairing.

[0067] FIG. 33 is a CrossmAb which is a heterodimeric construct with 2 different target binding Fabs 1 and 2 fused to a heterodimerization stabilized Fc. The CL and CH1 domains and the VH and VL domains switch, for example, CH1 merges in line with VL while CL merges in line with VH.

[0068] FIG. 34 is Fit-Ig, which are homodimeric constructs in which a Fab that binds antigen 2 is fused to the N-terminus of an HC Fab that binds antigen 1. The construct contains wild-type Fc.

[0069] FIG. 35 shows the binding of TriNKETs, which include a CAIX binding domain and a NKG2D binding domain, to NKG2D expressed on EL4 cells.

[0070] FIG. 36 shows binding of TriNKETs, which include a CAIX binding domain and a NKG2D binding domain, to CAIX expressed on A498 human renal cell carcinoma cells.

[0071] FIG. 37A shows TriNKET-mediated cytotoxicity of KHYG-1 effector cells to CAIX-positive A498 renal cell carcinoma cells. The dotted line indicates background killing of A498 cells during co-cultivation with resting KHYG-1 cells without TriNKET addition. FIG. 37B shows TriNKET-mediated cytotoxicity of resting NK cells to CAIX-positive A498 renal carcinoma cells.

DETAILED DESCRIPTION

[0072] The invention relates to polyspecific binding proteins that bind to CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 on a tumor cell and to the NKG2D receptor and CD16 receptor on natural killer cells to activate natural killer cells, to pharmaceutical compositions containing such polyspecific binding proteins, and therapeutic methods using such polyspecific proteins and pharmaceutical compositions, including for the treatment of malignant neoplasm. Various aspects of the invention are set forth in the sections below; however, aspects of the invention described in one particular section should not be limited to any particular section.

[0073] To facilitate understanding of the present invention, a number of terms and expressions are defined below.

[0074] The term in the singular as used herein means "one or more" and includes the plural unless the context is inappropriate. As used herein, the term "antigen binding site" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. In human antibodies, the antigen-binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. The three highly divergent regions in the V regions of the heavy and light chains are referred to as "hypervariable regions", which lie between more conserved flanking regions known as "framework regions" or "FRs". Thus, the term "FR" refers to amino acid sequences that naturally occur between and adjacent to hypervariable regions in immunoglobulins. In a human antibody molecule, three light chain hypervariable regions and three heavy chain hypervariable regions are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs". In some animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain, forming a "single domain antibody". Antigen-binding sites may exist in an intact antibody, in an antigen-binding fragment of an antibody that retains an antigen-binding surface, or in a recombinant polypeptide such as scFv using a peptide linker to connect a heavy chain variable domain to a light chain variable domain in a single polypeptide.

[0075] As used herein, the term "tumor-specific antigen" means any antigen, including, but not limited to, protein, glycoprotein, ganglioside, carbohydrate, lipid, that is associated with cancer. Such an antigen may be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates.

[0076] As used herein, the terms "subject" and "patient" refer to an organism to be treated with the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (eg, mice, monkeys, horses, cattle, pigs, dogs, cats, and the like) and more preferably include humans.

[0077] As used herein, the term "effective amount" refers to an amount of a compound (eg, a compound of the present invention) sufficient to achieve beneficial or desired results. An effective amount may be administered in one or more administrations, uses, or dosages and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treatment" includes any effect, such as reduction, reduction, modulation, amelioration, or elimination, that results in an improvement in a condition, disease, disorder, or the like. or amelioration of its symptom.

[0078] Used in the present description, the term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, which makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0079] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers such as phosphate-buffered saline, water, emulsions (such as oil/water or water/oil emulsions), and various types of wetting agents. agents. The compositions may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA [1975].

[0080] As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention that, when administered to a patient, is capable of providing the compound of the present invention or the active metabolite or residue thereof. As will be known to those skilled in the art, "salts" of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, chloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic , benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid and the like. Other acids such as oxalic acid, although not pharmaceutically acceptable per se, may be used in the preparation of salts useful as intermediates in the preparation of the compounds of the invention and their pharmaceutically acceptable acid addition salts.

[0081] Examples of bases include, but are not limited to, alkali metal (eg sodium) hydroxides, alkaline earth metal (eg magnesium) hydroxides, ammonia, and compounds of the formula NW4+ where W is C1-4 alkyl, and the like.

[0082] Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate and the like. Other examples of salts include anions of the compounds of the present invention coupled with a suitable cation such as Na+, NH4+ and NW4+ (where W is a C1-4 alkyl group) and the like.

[0083] For therapeutic use, salts of the compounds of the present invention are considered to be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0084] Throughout the specification, where compositions are described as having, including, or containing specific components, or where processes and methods are described as having, including, or containing specific steps, it is contemplated that there are additionally compositions of the present invention that consist essentially of, or consist of of the listed components and that there are processes and methods according to the present invention that consist essentially or consist of the listed processing steps.

[0085] As a rule, if a percentage is indicated in the composition, then this is a percentage by weight, unless otherwise indicated. Also, if a variable is not followed by a definition, then the previous definition of the variable takes precedence.

I. Proteins

[0086] The invention relates to polyspecific binding proteins that bind to CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 on a tumor cell and the NKG2D receptor and CD16 receptor on natural killer cells to activate the natural killer cell. Multispecific binding proteins are useful in the pharmaceutical compositions and therapeutic methods described herein. Binding of the polyspecific binding protein to the NKG2D receptor and the CD16 receptor on the natural killer cell enhances the activity of the natural killer cell to destroy the tumor cell. Binding of the polyspecific binding protein to CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 on the tumor cell causes the tumor cell to approach the natural killer cell, which facilitates direct and indirect destruction of the tumor cell by the natural killer cell. Further description of examples of polyspecific binding proteins is given below.

[0087] The first component of the polyspecific binding proteins binds to cells expressing the NKG2D receptor, which may include, but are not limited to, NK cells, γδ T cells, and CD8+ αβ T cells. With NKG2D binding, multispecific binding proteins can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NKG2D receptors.

[0088] The second component of the multispecific binding proteins binds to cells expressing CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2. CAIX expressing cells include, but are not limited to, renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, tumors of the esophagus, lung, colon, kidney, cervical, and non-small cell lung carcinoma. Mesothelin-expressing cells include, but are not limited to, mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, cholangiocarcinoma, gastric cancer, uterine serous carcinoma, thymus carcinoma, and acute myeloid leukemia. ANO1-expressing cells include, but are not limited to, esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumor (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, breast cancer, prostate cancer, and sarcoma. TROP2-expressing cells include, but are not limited to, breast, lung, stomach, colorectal, pancreas, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma, and ovarian carcinoma. CEA-expressing cells include, but are not limited to, gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. Claudin-18.2-expressing cells include, but are not limited to, esophageal cancer, non-small cell lung carcinoma, ovarian cancer, colon cancer, and several forms of bile duct carcinoma.

[0089] The third component for polyspecific binding proteins binds to cells expressing CD16, the Fc receptor on the surface of leukocytes, including natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells.

[0090] The polyspecific binding proteins described herein can take a variety of formats. For example, one format is a heterodimeric polyspecific antibody that includes a first immunoglobulin heavy chain, a first immunoglobulin light chain, and a second immunoglobulin heavy chain and a second immunoglobulin light chain (FIG. 1). The first immunoglobulin heavy chain includes a first Fc domain (hinge-CH2-CH3), a first heavy chain variable domain, and an optional first heavy chain CH1 domain. The first immunoglobulin light chain includes a first light chain variable domain and a first light chain constant domain. The first immunoglobulin light chain together with the first immunoglobulin heavy chain forms an antigen-binding site that binds to NKG2D. The second immunoglobulin heavy chain contains a second Fc domain (hinge-CH2-CH3), a second heavy chain variable domain, and optionally a second heavy chain CH1 domain. The second immunoglobulin light chain includes a second light chain variable domain and a second light chain constant domain. The second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen-binding site that binds to CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2. The first Fc domain and the second Fc domain together can bind to CD16 (FIG. 1). In some embodiments, the first immunoglobulin light chain is identical to the second immunoglobulin light chain.

[0091] Another exemplary format includes a heterodimeric polyspecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (FIG. 2). The first heavy chain of an immunoglobulin comprises a first Fc domain (hinge-CH2-CH3) fused, either through a linker or through an antibody hinge, to a single chain variable fragment (scFv) consisting of a heavy chain variable domain and a light chain variable domain that are paired and bind to NKG2D or bind to one of the tumor-specific antigens selected from CAIX, ANO1, mesothelin, TROP2, CEA and Claudin-18.2. The second immunoglobulin heavy chain includes a second Fc domain (hinge-CH2-CH3), a second heavy chain variable domain, and optionally a CH1 heavy chain domain. The light chain of an immunoglobulin comprises a light chain variable domain and a light chain constant domain. The second immunoglobulin heavy chain couples to the immunoglobulin light chain and binds to NKG2D or binds to one of the tumor specific antigens selected from CAIX, ANO1, mesothelin, TROP2, CEA and Claudin-18.2. The first Fc domain and the second Fc domain together can bind to CD16 (FIG. 2).

[0092] One or more additional binding motifs may be fused to the C-terminus of the CH3 domain of the constant region, optionally via a linker sequence. In some embodiments, the antigen binding site may be a single chain or disulfide chain stabilized variable region (scFv), or may form a tetravalent or trivalent molecule.

[0093] In some embodiments, the polyspecific binding protein is in the form of Triomab, which is a trifunctional bispecific antibody that maintains an IgG-like form. This chimera consists of two half antibodies, each with one light and one heavy chain, which are derived from two parental antibodies.

[0094] In some embodiments, the polyspecific binding protein is a common light chain (LC) form of KiH that utilizes a bump into a hole (KIH) technique. The KIH includes engineered CH3 domains to create either a "knob" or "trough" in each heavy chain to promote heterodimerization. The concept of the Fc ridge-in-the-pit (KiH) technology was to introduce a ridge in one CH3 domain (CH3A) by replacing a small residue with a bulky one (eg, T366WCH3A in EU numbering). To accommodate the ridge, a complementary 'trough' surface was created on the other CH3 domain (CH3B) by replacing the residues closest to the ridge with smaller residues (ie, T366S/L368A/Y407VCH3B). The trough mutation was optimized by structure-driven phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. Biol. (1997) 270(1 ):26-35). X-ray crystal structures of KiH Fc variants (Elliott JM, Ultsch M, Lee J, Tong R, Takeda K, Spiess C, et al., Antiparallel conformation of knob and hole aglycosylated half-antibody homodimers is mediated by a CH2-CH3 hydrophobic interaction. J Mol Biol (2014) 426(9):1947-57 Mimoto F, Kadono S, Katada H, Igawa T, Kamikawa T, Hattori K. Crystal structure of a novel asymmetrically engineered Fc variant with improved affinity for FcgammaRs. Mol. Immunol. (2014) 58 (1): 132-8) demonstrated that heterodimerization is thermodynamically supported by hydrophobic interactions mediated by steric complementarity at the core interface between CH3 domains, while the ridge-knob and trough-trough interfaces do not favor homodimerization due to steric interference and disruption of favorable interactions, respectively.

[0095] In some embodiments, the polyspecific binding protein is in the form of a dual variable domain immunoglobulin (DVD-Ig™) that combines the binding domains of the targets of two monoclonal antibodies through flexible natural linkers to form a tetravalent IgG-like molecule.

[0096] In some embodiments, the polyspecific binding protein is in the form of an orthogonal Fab interface (Ortho-Fab). In the ortho-Fab IgG approach (Lewis SM, Wu X, Pustilnik A, Sereno A, Huang F, Rick HL, et al., Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat. Biotechnol. ( 2014) 32 (2): 191-8), the structure-based regional design introduces complementary mutations at the LC and HCVH-CH1 interface in only one Fab without any change in the other Fab.

[0097] In some embodiments, the polyspecific binding protein is in a 2-in-1 Ig format. In some embodiments, the polyspecific binding protein is in the form of an ES, which is a heterodimeric construct containing 2 different Target 1 and Target 2 binding Fabs fused to an Fc. Heterodimerization is mediated by electrostatic interaction mutations in Fc.

[0098] In some embodiments, the polyspecific binding protein is in the form of a κλ-Body, which is heterodimeric constructs with 2 different Fab fused to Fc stabilized by heterodimerization mutations: Fab1 targeting antigen 1 contains kappa-LC, while the second Fab targeting antigen 2 contains lambda LC. FIG. 30A is an exemplary representation of one form of ĸλ-Body; FIG. 30B is an exemplary representation of another ĸλ-Body.

[0099] In some embodiments, the polyspecific binding protein is in a Fab exchange form (antibodies that exchange Fab fragments by replacing a heavy chain and an attached light chain (half-molecule) with a heavy-light chain pair from another molecule, such that leads to the production of bispecific antibodies).

[00100] In some embodiments, the polyspecific binding protein is in the form of a SEED Body. The engineered strand exchange domain (SEED) platform has been designed to generate asymmetric and bispecific antibody-like molecules, thus expanding the therapeutic applications of natural antibodies. This protein-engineered platform is based on the exchange of structurally related immunoglobulin sequences in conserved CH3 domains. The SEED construct allows efficient generation of AG/GA heterodimers while preventing homodimerization of the AG and GA domains of SEED CH3. (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5): 447-54).

[00101] In some embodiments, the implementation of the polyspecific binding protein is in the form of LuZ-Y, in which the domain of the leucine zipper is used to induce heterodimerization of two different HC. (Wranik, BJ. et al., J. Biol. Chem. (2012), 287: 43331-9).

[00102] In some embodiments, the polyspecific binding protein is in the form of a Cov-X-Body. In bispecific CovX-Body, two different peptides are linked together using a branched azetidinone linker and fused to the scaffold antibody under mild conditions in a site-specific manner. While pharmacophores are responsible for functional activity, the antibody scaffold provides a long half-life and Ig-like distribution. Pharmacophores can be chemically optimized or replaced with other pharmacophores to produce optimized or unique bispecific antibodies. (Doppalapudi VR et al., PNAS (2010), 107 (52); 22611-22616).

[00103] In some embodiments, the polyspecific binding protein is in the heterodimeric form of Oasc-Fab, which includes a Fab that binds to target 1 and a scFab that binds to target 2 fused to an Fc. Heterodimerization is provided by mutations in Fc.

[00104] In some embodiments, the multispecific binding protein is in the form of a DuetMab, which is a heterodimeric construct containing 2 different Antigen-binding Fabs 1 and 2 and an Fc stabilized by heterodimerization mutations. Fab 1 and 2 contain differential SS bridges that ensure proper pairing of LC and HC.

[00105] In some embodiments, the multispecific binding protein is in the form of a CrossmAb, which is a heterodimeric construct with 2 different target-binding Fabs 1 and 2 fused to Fc stabilized by heterodimerization. The CL and CH1 domains and the VH and VL domains switch, for example, CH1 merges in line with VL while CL merges in line with VH.

[00106] In some embodiments, the polyspecific binding protein is in the form of Fit-Ig, which are homodimeric constructs where the Fab that binds to antigen 2 is fused to the N-terminus of the HC Fab that binds to antigen 1. The construct contains a wild-type Fc .

[00107] Table 1 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to NKG2D. NKG2D binding domains can vary in their binding affinity for NKG2D, however, they all activate human NKG2D and NK cells.

Table 1 clones Amino acid sequence of the heavy chain variable region Amino acid sequence of the light chain variable region ADI-27705 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:1)
CDR1 (SEQ ID NO:51)-GSFSGYYWS
CDR2 (SEQ ID NO:52) -
EIDHSSGSTNYNPSLKS
CDR3 (SEQ ID NO:53) -
ARARGPWSFDP DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPITFGGGTKVEIK
(SEQ ID NO:2) ADI-27724 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:3) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYCQQYGSSPITFGGGTKVEIK
(SEQ ID NO:4) ADI-27740 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:5) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSFYTFGGGTKVEIK
(SEQ ID NO:6) ADI-27741 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:7) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQSNSYYTFGGGTKVEIK
(SEQ ID NO:8) ADI-27743 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:9) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPTFGGGTKVEIK
(SEQ ID NO:10) ADI-28153 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWGFDPWGQGTLVTVSS
(SEQ ID NO:11) ELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLEIK
(SEQ ID NO:12) ADI-28226 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:13) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYGSFPITFGGGTKVEIK
(SEQ ID NO:14) ADI-28154 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:15) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK
(SEQ ID NO:16) ADI-29399 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:17) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSFPTFGGGTKVEIK
(SEQ ID NO:18) ADI-29401 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:19) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDIYPTFGGGTKVEIK
(SEQ ID NO:20) ADI-29403 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:21) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDSYPTFGGGTKVEIK
(SEQ ID NO:22) ADI-29405 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:23) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYGSFPTFGGGTKVEIK
(SEQ ID NO:24) ADI-29407 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:25) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYQSFTFGGGTKVEIK
(SEQ ID NO:26) ADI-29419 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:27) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYSSFSTFGGGTKVEIK
(SEQ ID NO:28) ADI-29421 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:29) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYESYSTFGGGTKVEIK
(SEQ ID NO:30) ADI-29424 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:31) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDSFITFGGGTKVEIK
(SEQ ID NO:32) ADI-29425 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:33) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYQSYPTFGGGTKVEIK
(SEQ ID NO:34) ADI-29426 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:35) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSFPTFGGGTKVEIK
(SEQ ID NO:36) ADI-29429 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:37) DIQMTQSPSTLSASVGDRVTITCRASQSIGSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYELYSYTFGGGTKVEIK
(SEQ ID NO:38) ADI-29447 QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:39) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDTFITFGGGTKVEIK
(SEQ ID NO:40) ADI-27727 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGDSSIRHAYYYYGMDVWGQGTTVTVSS
(SEQ ID NO:41)
CDR1 (SEQ ID NO:54) -
GTFSSYAIS
CDR2 (SEQ ID NO:55) -
GIIPIFGTANYAQKFQG
CDR3 (SEQ ID NO:56) -
ARGDSSIRHAYYYYGMDV DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPITFGGGTKVEIK
(SEQ ID NO:42)
CDR1 (SEQ ID NO:57) -
KSSQSVLYSSNNKNYLA
CDR2 (SEQ ID NO:58) -
WASTRES
CDR3 (SEQ ID NO:59) -
QQYYSTPIT ADI-29443 QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGSDRFHPYFDYWGQGTLVTVSS
(SEQ ID NO:43)
CDR1 (SEQ ID NO:60) -
GSISSSSYYWG
CDR2 (SEQ ID NO:61) -
SIYYSGSTYYNPSLKS
CDR3 (SEQ ID NO:62) -
ARGSDRFHPYFDY EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQFDTWPPTFGGGTKVEIK
(SEQ ID NO:44)
CDR1 (SEQ ID NO:63) -
RASQSVSRYLA
CDR2 (SEQ ID NO:64) -
DASNRAT
CDR3 (SEQ ID NO:65) -
QQFDTWPPT ADI-28200 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRGRKASGSFYYYYGMDVWGQGTTVTVSS
(SEQ ID NO:45)
CDR1 (SEQ ID NO:66) - GTFSSYAIS
CDR2 (SEQ ID NO:67) - GIIPIFGTANYAQKFQG
CDR3 (SEQ ID NO:68) - ARRGRKASGSFYYYYGMDV DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK
(SEQ ID NO:46)
CDR1 (SEQ ID NO:69) - ESSQSLLNSGNQKNYLT
CDR2 (SEQ ID NO:70) - WASTRES
CDR3 (SEQ ID NO:71) - QNDYSYPYT ADI-27744
(A44) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGGYYDSGAGDYWGQGTLVTVSS
(SEQ ID NO:132)
CDR1 (SEQ ID NO:134) - FTFSSYAMS
CDR2 (SEQ ID NO:135) - AISGSGGSTYYADSVKG
CDR3 (SEQ ID NO:136) - AKDGGYYDSGAGDY DIQMTQSPSSVSASVGDRVTITCRASQGIDSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSYPRTFGGGTKVEIK
(SEQ ID NO:133)
CDR1 (SEQ ID NO:137) - RASQGIDSWLA
CDR2 (SEQ ID NO:138) - AASSLQS
CDR3 (SEQ ID NO:139) - QQGVSYPRT ADI-27749
(A49) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPMGAAAGWFDPWGQGTLVTVSS
(SEQ ID NO:140)
CDR1 (SEQ ID NO:142) - FTFSSYSMN
CDR2 (SEQ ID NO:143) - SISSSSSYIYYADSVKG
CDR3 (SEQ ID NO:144) - ARGAPMGAAAGWFDP DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIK
(SEQ ID NO:141)
CDR1 (SEQ ID NO:145) - RASQGISSWLA
CDR2 (SEQ ID NO:146) - AASSLQS
CDR3 (SEQ ID NO:147) - QQGVSFPRT ADI-29463
(F63) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDTGEYYDTDDHGMDVWGQGTTVTVSS
(SEQ ID NO:148)
CDR1 (SEQ ID NO:150) - YTFTGYYMH
CDR2 (SEQ ID NO:151) - WINPNSGGTNYAQKFQG
CDR3 (SEQ ID NO:152) - ARDTGEYYDTTDDHGMDV EIVLTQSPGTLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDDYWPPTFGGGTKVEIK
(SEQ ID NO:149)
CDR1 (SEQ ID NO:153) - RASQSVSSNLA
CDR2 (SEQ ID NO:154) - GASTRAT
CDR3 (SEQ ID NO:155) - QQDDYWPPT ADI-29379
(E79) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGAPNYGDTTHDYYYMDVWGKGTTVTVSS
(SEQ ID NO:156)
CDR1 (SEQ ID NO:158) - YTFTSYYMH
CDR2 (SEQ ID NO:159) - IINPSGGSTSYAQKFQG
CDR3 (SEQ ID NO:160) - ARGAPNYGDTTHDYYYMDV EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDDWPFTFGGGTKVEIK
(SEQ ID NO:157)
CDR1 (SEQ ID NO:161) - RASQSVSSNLA
CDR2 (SEQ ID NO:162) - GASTRAT
CDR3 (SEQ ID NO:163) - QQYDDWPFT ADI-29447
(F47) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEIDHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARARGPWSFDPWGQGTLVTVSS
(SEQ ID NO:164)
CDR1 (SEQ ID NO:166) - GSFSGYYWS
CDR2 (SEQ ID NO:167) - EIDHSGSTNYNPSLKS
CDR3 (SEQ ID NO:168) - ARARGPWSFDP DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDTFITFGGGTKVEIK
(SEQ ID NO:165)
CDR1 (SEQ ID NO:169) - RASQSISSWLA
CDR2 (SEQ ID NO:170) - KASSLES
CDR3 (SEQ ID NO:171) - QQYDTFIT

[00108] Alternatively, the heavy chain variable domain defined by SEQ ID NO: 47 can be paired with the light chain variable domain defined by SEQ ID NO: 48 to form an antigen binding site that can bind to NKG2D, as illustrated in US Pat. No. 9,273,136.

SEQ ID NO:47

SEQ ID NO:75:

QSALTQPASVSGSPGQSITISCSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSAFLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL

[00109] Alternatively, the heavy chain variable domain defined by SEQ ID NO: 49 can be paired with the light chain variable domain defined by SEQ ID NO: 50 to form an antigen binding site that can bind to NKG2D, as illustrated in US Patent 7,879,985.

SEQ ID NO:49

QVHLQESGPGLVKPSETLSLTCTVSDDSISSYYWSWIRQPPGKGLEWIGHISYSGS ANYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCANWDDAFNIWGQGTMVTVSS

SEQ ID NO:50

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK

[00110] Table 2 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to CAIX.

table 2 clones Amino acid sequence of the heavy chain variable domain Amino acid sequence of the light chain variable domain Girentuximab (PCT/EP2002/001283) DVKLVESGGGLVKLGGSLKLSCAASGFTFSNYYMSWVRQTPEKRLELVAAINSDGGITYYLDTVKGRFTISRDNAKNTLYLQMSSLKSEDTALFYCARHRSGYFSMDYWGQGTSVTVSS
(SEQ ID NO:72)
CDR1 (SEQ ID NO:74) - GFTFSNY
CDR2 (SEQ ID NO:75) - NSDGGI
CDR3 (SEQ ID NO:76) - HRSGYFSMDY DIVMTQSQRFMSTTVGDRVSITCKASQNVVSAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNMQSEDLADFFCQQYSNYPWTFGGGTKLEIKR
(SEQ ID NO:73)
CDR1(SEQ ID NO:77)-QNVVSAVA
CDR2 (SEQ ID NO:78) - SASNRYT
CDR3 (SEQ ID NO:79) - QQYSNYPWT BAY79-4620
(US 8168758) QVELVESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSGISSLGSTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTGSPGTFMHGDHWGQGTLVTVSS (SEQ ID NO:80)
CDR1 (SEQ ID NO:82) - GFTFSSYGMS
CDR2 (SEQ ID NO:83) - GISSLGSTTYYADSVKG
CDR3 (SEQ ID NO:84) - TGSPGTFMHGDH DIQMTQSPSSLSASVGDRVTITCRASQDINNYLSWYQQKPGKAPKLLIYGASNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQYYGRPTTFGQGTKVEIK
(SEQ ID NO:81)
CDR1 (SEQ ID NO:85) - RASQDINNYLS
CDR2 (SEQ ID NO:86) - YGASNLQS
CDR3 (SEQ ID NO:87) - QQYYGRPT anti-CAIX
(US 12/016884) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGQRLELVSAINSDGGITYYLDSVKGRFTISRDNAKNTLYLQMGSLRAEDMAVYYCARHRSGYFSMDYWGQGTLVTVSS
(SEQ ID NO:88)
CDR1 (SEQ ID NO:90) - GFTFSNY
CDR2 (SEQ ID NO:91) - NSDGGI
CDR3 (SEQ ID NO:92) - HRSGYFSMDY DIVMTQSPSSLSASVGDRVTITCRASQNVVSAVAWYLQKPGKSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYFCQQYSNYPWTFGGGTKLEIK
(SEQ ID NO:89)
CDR1 (SEQ ID NO:93) - QNVVSAVA
CDR2 (SEQ ID NO:94) - SASNRYT
CDR3 (SEQ ID NO:95) - QQYSNYPWT

[00111] Alternatively, novel antigen-binding sites that bind to CAIX can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 96.

SEQ ID NO:96

MAPLCPSPWLPLLIPAPAPGLTVQLLLSLLLLVPVHPQRLPRMQEDSPLGGGSSGE DDPLGEEDLPSEEDSPREEDPPGEEDLPGEEDLPGEEDLPEVKPKSEEEGSLKLEDLPTVE APGDPQEPQNNAHRDKEGDDQSHWRYGGDPPWPRVSPACAGRFQSPVDIRPQLAAFCP ALRPLELLGFQLPPLPELRLRNNGHSVQLTLPPGLEMALGPGREYRALQLHLHWGAAGR PGSEHTVEGHRFPAEIHVVHLSTAFARVDEALGRPGGLAVLAAFLEEGPEENSAYEQLLS RLEEIAEEGSETQVPGLDISALLPSDFSRYFQYEGSLTTPPCAQGVIWTVFNQTVMLSAK QLHTLSDTLWGPGDSRLQLNFRATQPLNGRVIEASFPAGVDSSPRAAEPVQLNSCLAAG DILALVFGLLFAVTSVAFLVQMRRQHRRGTKGGVSYRPAEVAETGA

[00112] Table 3 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to ANO1.

Table 3 clones Amino acid sequence of the heavy chain variable domain Amino acid sequence of the light chain variable domain ANO1 antibody (PCT/US2012/067430) QVQLKESGPGLVQPSQTLTLTCTVSGFSLSNYDMHWVRQSPGKGLDWMGVIWGNGKTQYNSGLTSRLSISRDTSKSQVFLKMNSLQTEDTAIYFCTRSGYYYDGSYYSLFDYWGQGVMVTVSS
(SEQ ID NO:97)
CDR1 (SEQ ID NO:99) - NYDMH
CDR2 (SEQ ID NO:100) - VIWGNGKTQYNSGLTS
CDR3 (SEQ ID NO:101) - SGYYYDGSYYSLFDY DVVLTQTPPTLSATIGQSVSISCRSSQSLLHSNGNTYLNWLLQRPGQPPQLLIYLVSRLESGVPNRFSGSGSGTDFTLKISGVEAEDLGVYYCVQSTHAPAFGGGTKLELK
(SEQ ID NO:98)
CDR1(SEQ ID NO:102)-RSSQSLLHSNGNTYLN
CDR2 (SEQ ID NO:103) - LVSRLES
CDR3 (SEQ ID NO:104) - VQSTHAPA

[00113] Alternatively, novel antigen-binding sites that bind to ANO1 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 105.

SEQ ID NO:105

MRVNEKYSTLPAEDRSVHIINICAIEDIGYLPSEGTLLNSLSVDPDAECKYGLYFR DGRRKVDYILVYHHKRPSGNRTLVRRVQHSDTPSGARSVKQDHPLPGKGASLDAGSGEPPMDYHEDDKRFRREEYEGNLLEAGLELERDEDTKIHGVGFVKIHAPWNVLCREAEFLKLKMPTKKMYHINETRGLLKKINSVLQKITDPIQPKVAEHRPQTMKRLSYPFSREKQHLFDLSDKDSFFDSKTRSTIVYEILKRTTCTKAKYSMGITSLLANGVYAAAYPLHDGDYNGENVEFNDRKLLYEEWARYGVFYKYQPIDLVRKYFGEKIGLYFAWLGVYTQMLIPASIVGIIVFLYGCATMDENIPSMEMCDQRHNITMCPLCDKTCSYWKMSSACATARASHLFDNPATVFFSVFMALWAATFMEHWKRKQMRLNYRWDLTGFEEEEEAVKDHPRAEYEARVLEKSLKKESRNKEKRRHIPEESTNKWKQRVKTAMAGVKLTDKVKLTWRDRFPAYLTNLVSIIFMIAVTFAIVLGVIIYRISMAAALAMNSSPSVRSNIRVTVTATAVIINLVVIILLDEVYGCIARWLTKIEVPKTEKSFEERLIFKAFLLKFVNSYTPIFYVAFFKGRFVGRPGDYVYIFRSFRMEECAPGGCLMELCIQLSIIMLGKQLIQNNLFEIGIPKMKKLIRYLKLKQQSPPDHEECVKRKQRYEVDYNLEPFAGLTPEYMEMIIQFGFVTLFVASFPLAPLFALLNNIIEIRLDAKKFVTELRRPVAVRAKDIGIWYNILRGIGKLAVIINAFVISFTSDFIPRLVYLYMYSKNGTMHGFVNHTLSSFNVSDFQNGTAPNDPLDLGYEVQICRYKDYREPPWSENKYDISKDFWAVLAARLAFVIVFQNLVMFMSDFVDWVIPDIPKDISQQIHKEKVLMVELFMREEQDKQQLLETWMEKERQKDEPPCNHHNTKACPDSLGSPAPSHAYHGGVL

[00114] Table 4 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to mesothelin.

Table 4 Amino acid sequence of the heavy chain variable domain Amino acid sequence of the light chain variable domain clones Heavy chain variable domain amino acid sequence Light chain variable domain amino acid sequence Amatuximab (US 7592426) QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSA
(SEQ ID NO:106)
CDR1 (SEQ ID NO:108) - GYSFTGY
CDR2 (SEQ ID NO:109) - TPYNGA
CDR3 (SEQ ID NO:110) - GGYDGRGFDY DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKR
(SEQ ID NO:107)
CDR1(SEQ ID NO:111)-SSVSYMH
CDR2 (SEQ ID NO:112) - DTSKLAS
CDR3 (SEQ ID NO:113) - QQWSKHPLT anti-mesothelin
(US 8425904) EVHLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAQGKGLEWVASIKQAGSEKTYVDSVKGRFTISRDNAKNSLSLQMNSLRAEDTAVYYCAREGAYYYDSASYYPYYYYYSMDVWGQGTTVTVSS
(SEQ ID NO:114)
CDR1 (SEQ ID NO:116) - GFTFSRY
CDR2 (SEQ ID NO:117) - KQAGSE
CDR3 (SEQ ID NO:118) - EGAYYYDSASYYPYYYYYSMDV EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYCQQYGSSQYTFGQGTKLEIK
(SEQ ID NO:115)
CDR1 (SEQ ID NO:119) - QSVSSSYLA
CDR2 (SEQ ID NO:120) - GASSRAT
CDR3 (SEQ ID NO:121) - QQYGSSQYT Anetumab
(US 9023351) QVELVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSSA
(SEQ ID NO:122)
CDR1 (SEQ ID NO:124) - GYSFTSY
CDR2 (SEQ ID NO:125) - DPGDSR
CDR3 (SEQ ID NO:126) - GQLYGGTYMDG DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLTVLG
(SEQ ID NO:123)
CDR1 (SEQ ID NO:127) - SSDIGGYNSVS
CDR2 (SEQ ID NO:128) - GVNNRPS
CDR3 (SEQ ID NO:129) - SSYDIESATPV

[00115] Alternatively, novel antigen-binding sites that bind to mesothelin can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 130.

SEQ ID NO:130

MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANP PNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA

[00116] Table 5 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to TROP2.

Table 5 clones Amino acid sequence of the heavy chain variable domain Amino acid sequence of the light chain variable domain IMMU-132 (US 7238785) SVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS
(SEQ ID NO:172)
CDR1 (SEQ ID NO:174) - GYTFTNY
CDR2 (SEQ ID NO:175) - NTYTGE
CDR3 (SEQ ID NO:176) - GGFGSSYWYFDV DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKR
(SEQ ID NO:173)
CDR1(SEQ ID NO:177)-QDVSIAVA
CDR2 (SEQ ID NO:178) - SASYRYT
CDR3 (SEQ ID NO:179) - QQHYITPLT anti-TROP2 antibody
(PCT/CA08/000979) QIQLVQSGHEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTKTGEPTYAEEFKGRFVFSLETSASTAYLQISSLKAEDTAMYFCGRGGYGSSYWYFDVWGQGTTVTVSS
(SEQ ID NO:180)
CDR1 (SEQ ID NO:182) - GYTFTNY
CDR2 (SEQ ID NO:183) - NTKTGE
CDR3 (SEQ ID NO:184) - GGYGSSYWYFDV DIQMTQSPSSLSASVGDRVTITCKASQDVSIAVAWYQQKPGKAPKVLIYSASYRYTGVPDRFSGSGSGTDFTFTISSLQPEDIAVYYCQQHYITPLTFGGGTKVEIK
(SEQ ID NO:181)
CDR1 (SEQ ID NO:185) - QDVSIAVA
CDR2 (SEQ ID NO:186) - SASYRYT
CDR3 (SEQ ID NO:187) - QQHYITPLT anti-TROP2 antibody
(US15/187179) QVQLVQSGAEVKKPGASVKVSCKASGYTFTTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISADTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS
(SEQ ID NO:188)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTTTAGMQWVRQAPGQGLEWMGWINTHSGVPKYAEDFKGRVTISLDTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS
(SEQ ID NO:190)
QIQLVQSGAEVKKPGESVKVSCKASGYTFTTTAGMQWVQQMPGKGLEWMGWINTHSGVPKYAEDFKGRVTFSLDTSTSTAYLQLSSLKSEDTAVYYCARSGFGSSYWYFDVWGQGTLVTVSS
(SEQ ID NO:192) DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRT
(SEQ ID NO:189)
DIVMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKRT
(SEQ ID NO:191)
DIVMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKQPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGQGTKLEIKR
(SEQ ID NO:193)

[00117] Alternatively, novel antigen-binding sites that bind to TROP2 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 194.

SEQ ID NO:194

MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVRTHHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL

[00118] Table 6 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to CEA.

Table 6 clones Amino acid sequence of the heavy chain variable domain Amino acid sequence of the light chain variable domain labetuzumab EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS
(SEQ ID NO:195)
CDR1 (SEQ ID NO:197) - GFDFTTY
CDR2 (SEQ ID NO:198) - HPDSST
CDR3 (SEQ ID NO:199) - LYFGFPWFAY DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK
(SEQ ID NO:196)
CDR1(SEQ ID NO:200)-QDVGTSVA
CDR2 (SEQ ID NO:201) - WTSTRHT
CDR3 (SEQ ID NO:202) - QQYSLYRS cergutuzumab QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYVEAMDYWGQGTTVTVSSA
(SEQ ID NO:203)
CDR1 (SEQ ID NO:205) - GYTFTEF
CDR2 (SEQ ID NO:206) - NTKTGE
CDR3 (SEQ ID NO:207) - WDFAYYVEAMDY DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKLEIKR
(SEQ ID NO:204)
CDR1 (SEQ ID NO:208) - AAVGTYVA
CDR2 (SEQ ID NO:209) - SASYRKR
CDR3 (SEQ ID NO:210) - HQYYTYPLFT Anti-CEA
(US 9056911) QVQLQQSGAELVRSGASIKLSCTASGFNIKHYYMHWVKQRPEQGLEWIGWINPENVDTEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNHYRYAGGGALDYWGQGTTVTVSSA
(SEQ ID NO:211)
CDR1 (SEQ ID NO:213) - GFNIKHY
CDR2 (SEQ ID NO:214) - NPENVD
CDR3 (SEQ ID NO:215) - YRYAGGGALDY DIQLTQSPAIMSASPGEKVTMTCSASSSVSYIHWYQQKSGTSPKRWVYDTSKLASGVPARFSGSGSGTSYSLTISTMEAEDAATYYCQQWNNNPYSFGGGTKVVIKTV
(SEQ ID NO:212)
CDR1 (SEQ ID NO:216) - SSVSYIH
CDR2 (SEQ ID NO:217) - DTSKLAS
CDR3 (SEQ ID NO:218) - QQWNNNPYS

[00119] Alternatively, novel antigen-binding sites that bind to CEA can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 219.

SEQ ID NO:219

MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI

[00120] Table 7 lists the peptide sequences of heavy chain variable domains and light chain variable domains that, in combination, can bind to Claudin-18.2.

Table 7 clones Amino acid sequence of the heavy chain variable domain Amino acid sequence of the light chain variable domain 1 (PCT/EP2015/058206) QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTNTGEPTY
AEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARLGFGNAMDYWGQGTSVTVSS
(SEQ ID NO:220)
CDR1 (SEQ ID NO:222) - GYTFTNYG
CDR2 (SEQ ID NO:223) - INNTTGEP
CDR3 (SEQ ID NO:224) - ARLGFGNAMDY DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTR
ESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELK
(SEQ ID NO:221)
CDR1(SEQ ID NO:225)-QSLLNSGNQKNY
CDR2 (SEQ ID NO:226) - WAS
CDR3 (SEQ ID NO:227) - QNDYSYPLT 2 (PCT/EP2015/058206) QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNY
NQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTRSWRGNSFDYWGQGTTLTVSS
(SEQ ID NO:228)
CDR1 (SEQ ID NO:230) - GYTFTSYW
CDR2 (SEQ ID NO:231) - IYPSDSYT
CDR3 (SEQ ID NO:232) - TRSWRGNSFDY DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTR
ESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK
(SEQ ID NO:229)
CDR1 (SEQ ID NO:233) - QSLLNSGNQKNY
CDR2 (SEQ ID NO:234) - WAS
CDR3 (SEQ ID NO:235) - QNDYSYPFT

[00121] Alternatively, novel antigen-binding sites that bind to Claudin-18.2 can be identified by screening for binding to the amino acid sequence defined by SEQ ID NO: 236.

SEQ ID NO:236

MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV

[00122] Within the Fc domain, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, interaction with CD16 is mainly focused on the amino acid residues Asp 265 - Glu 269, Asn 297 - Thr 299, Ala 327 - Ile 332, Leu 234 - Ser 239 and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see Sondermann et al., Nature, 406 (6793): 267-273). Based on known domains, mutations can be selected to increase or decrease binding affinity for CD16, for example using phage-exposed libraries or yeast surface-exposed cDNA libraries, or can be designed based on a known three-dimensional interaction pattern.

[00123] The assembly of heterodimeric antibody heavy chains can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which can result in homodimer assembly of each antibody heavy chain as well as heterodimer assembly. Stimulation of preferential assembly of heterodimers can be achieved by incorporating various mutations into the CH3 domain of each antibody heavy chain constant region, as shown in US13/494870, US16/028850, US11/533709, US12/875015, US13/289934, US14/773418, US12/ 811207, US13/866756, US14/647480, US14/830336. For example, mutations can be made in the CH3 domain of human IgG1 and include different pairs of amino acid substitutions in the first polypeptide and the second polypeptide that allow the two chains to selectively heterodimerize with each other. The positions of the amino acid substitutions shown below are numbered according to the EU index, as in Kabat.

[00124] In one scenario, an amino acid substitution in the first polypeptide replaces the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W) and at least one amino acid substitution in the second polypeptide replaces the original amino acid(s) with a smaller amino acid(s) selected from alanine (A), serine (S), threonine (T), or valine (V), so that the larger amino acid substitution (bulge) fits into the surface of the smaller amino acids replacement (cavity). For example, one polypeptide may include a T366W substitution and another may include three substitutions including T366S, L368A, and Y407V.

[00125] The heavy chain variable domain of an antibody of the invention may optionally be linked to an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region, including hinge domains, CH2 and CH3 domains with or without a CH1 domain . In some embodiments, the amino acid sequence of the constant region is at least 90% identical to a human antibody constant region, such as a human IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region. In some other embodiments, the amino acid sequence of the constant region is at least 90% identical to the constant region of an antibody from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations may be included in the constant region versus the constant region of human IgG1, e.g. , D399, S400, D401, F405, Y407, K409, T411 and/or K439. Typical replacements include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K34,W, S364KE S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I , Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E.

[00126] In some embodiments, mutations that may be included in the human IgG1 constant region CH1 may be at the amino acid V125, F126, P127, T135, T139, A140, F170, P171, and/or V173. In some embodiments, mutations that may be included in the Cκ human IgG1 constant region may be at the amino acid E123, F116, S176, V163, S174, and/or T164.

[00127] Alternatively, amino acid substitutions may be selected from the following sets of substitutions shown in Table 8.

Table 8 First polypeptide Second polypeptide Set 1 S364E/F405A Y349K/T394F Set 2 S364H/D401K Y349T/T411E Set 3 S364H/T394F Y349T/F405A Set 4 S364E/T394F Y349K/F405A Set 5 S364E/T411E Y349K/D401K Set 6 S364D/T394F Y349K/F405A Set 7 S364H/F405A Y349T/T394F Set 8 S364K/E357Q L368D/K370S Set 9 L368D/K370S S364K Set 10 L368E/K370S S364K Set 11 K360E/Q362E D401K Set 12 L368D/K370S S364K/E357L Set 13 K370S S364K/E357Q Set 14 F405L K409R Set 15 K409R F405L

[00128] Alternatively, amino acid substitutions may be selected from the following sets of substitutions shown in Table 9.

Table 9 First polypeptide Second polypeptide Set 1 K409W D399V/F405T Set 2 Y349S E357W Set 3 K360E Q347R Set 4 K360E/K409W Q347R/D399V/F405T Set 5 Q347E/K360E/K409W Q347R/D399V/F405T Set 6 Y349S/K409W E357W/D399V/F405T

[00129] Alternatively, amino acid substitutions may be selected from the following set of substitutions shown in Table 10.

Table 10 First polypeptide Second polypeptide Set 1 T366K/L351K L351D/L368E Set 2 T366K/L351K L351D/Y349E Set 3 T366K/L351K L351D/Y349D Set 4 T366K/L351K L351D/Y349E/L368E Set 5 T366K/L351K L351D/Y349D/L368E Set 6 E356K/D399K K392D/K409D

[00130] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 11.

Table 11 First polypeptide Second polypeptide L351Y, D399R, D399K, S400K, S400R, Y407A, Y407I, Y407V T366V, T366I, T366L, T366M, N390D, N390E, K392L, K392M, K392V, K392F K392D, K392E, K409F, K409W, T411D and T411E

[00131] Alternatively, at least one amino acid substitution may be selected from the following set of substitutions in Table 12, where the position(s) listed in the First Polypeptide column is replaced by any known negatively charged amino acid, and the position(s) indicated in the column "Second polypeptide", replaced by any known positively charged amino acid.

Table 12 First polypeptide Second polypeptide K392, K370, K409, or K439 D399, E356, or E357

[00132] Alternatively, at least one amino acid substitution may be selected from the following set in Table 13, where the position(s) listed in the "First Polypeptide" column is replaced by any known positively charged amino acid, and the position(s) indicated, indicated in the column "Second polypeptide", replaced by any known negatively charged amino acid.

Table 13 First polypeptide Second polypeptide D399, E356, or E357 K409, K439, K370, or K392

[00133] Alternatively, amino acid substitutions may be selected from the following set in Table 14.

Table 14 First polypeptide Second polypeptide T350V, L351Y, F405A, and Y407V T350V, T366L, K392L, and T394W

[00134] Alternatively or additionally, the structural stability of a heteromultimeric protein can be enhanced by introducing S354C on either the first or second polypeptide chain and Y349C on the opposite polypeptide chain that forms an artificial disulfide bridge within the interface of the two polypeptides.

[00135] The polyspecific proteins described above can be produced using recombinant DNA technology well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain may be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain may be cloned into a second expression vector; a third nucleic acid sequence encoding an immunoglobulin light chain can be cloned into a third expression vector; the first, second and third expression vectors can be stably transfected together into host cells to produce multimeric proteins.

[00136] In order to achieve the highest yield of a polyspecific protein, different ratios of the first, second, and third expression vector can be studied to determine the optimal ratio for transfection into host cells. After transfection, individual clones can be isolated to generate a cell bank using techniques known in the art such as limited dilution, ELISA, FACS, microscopy, or Clonepix.

[00137] Clones can be cultured under conditions suitable for scaling up for a bioreactor and maintaining the expression of a polyspecific protein. Multispecific proteins can be isolated and purified using methods known in the art, including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography.

II. Characterization of polyspecific proteins

[00138] In some embodiments, the polyspecific proteins described herein, which include an NKG2D binding domain and a binding domain for CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2, bind to cells expressing human NKG2D. In some embodiments, the polyspecific proteins bind to the CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 tumor-specific antigen at a level comparable to that of a monoclonal antibody having the same corresponding binding domain for CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin -18.2. However, the polyspecific proteins described herein may be more effective in reducing tumor growth and killing tumor cells expressing CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 than monoclonal antibodies corresponding to CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin - 18.2.

[00139] In some embodiments, polyspecific proteins described herein that include a NKG2D binding domain and a binding domain for CAIX, ANO1, mesothelin, TROP2, CEA, or Clauidin-18.2 can activate primary human NK cells when cultured with tumor cells expressing CAIX antigen, ANO1, mesothelin, TROP2, CEA or Claudin-18.2. Activation of NK cells is marked by an increase in CD107a degranulation and the production of IFNγ cytokines. In addition, when compared to a monoclonal antibody that includes the same CAIX-, ANO1-, mesothelin-, TROP2-, CEA-, or Claudin-18.2-binding domain, the polyspecific proteins show superior activation of human NK cells in the presence of tumor cells. expressing CAIX antigen, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2.

[00140] In some embodiments, polyspecific proteins described herein that include a NKG2D binding domain and a binding domain for CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 can enhance the activity of resting and IL-2-activated Human NK cells in the presence of tumor cells expressing CAIX antigen, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2.

[00141] In some embodiments, the polyspecific proteins described herein, which include a NKG2D binding domain and a binding domain for the tumor-specific antigen CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2, can enhance the cytotoxic activity of resting and IL- 2-activated human NK cells in the presence of tumor cells expressing CAIX antigen, ANO1, mesothelin, TROP2, CEA, or Clauidin-18.2. In some embodiments, compared to the corresponding monoclonal antibodies, polyspecific proteins can confer an advantage against tumor cells that express moderate to low levels of CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2.

[00142] In some embodiments, the polyspecific proteins described herein may be useful in the treatment of a highly expressed Fc receptor (FcR) cancer or a cancer in a tumor microenvironment with a high level of FcR compared to the corresponding CAIX monoclonal antibodies , ANO1, mesothelin, TROP2, CEA or Claudin-18.2. Monoclonal antibodies exert their influence on tumor growth through a variety of mechanisms, including but not limited to ADCC, CDC, phagocytosis, and signaling blockade. Among the FcγRs, CD16 has the lowest affinity for Fc IgG; FcγRI (CD64) is a high affinity FcR that binds about 1000 times more strongly to Fc IgG than CD16. CD64 is normally expressed on many cells of hematopoietic lineages, such as the myeloid lineage, and can be expressed on tumors derived from these cell types, such as acute myeloid leukemia (AML). Tumor infiltrating immune cells such as MDSC and monocytes also express CD64 and are known to infiltrate the tumor microenvironment. Expression of CD64 by the tumor or in the tumor microenvironment can have a detrimental effect on monoclonal antibody therapy. Expression of CD64 in the tumor microenvironment makes it difficult for these antibodies to interact with CD16 on the surface of NK cells, since the antibodies prefer to bind to the high affinity receptor. Multispecific proteins, by targeting two activating receptors on the surface of NK cells, can overcome the detrimental effect of CD64 expression (either on the tumor or the tumor microenvironment) on monoclonal antibody therapy. Regardless of CD64 expression on tumor cells, polyspecific proteins are able to mediate human NK cell responses against all tumor cells because dual targeting of the two activating receptors on NK cells provides stronger specific binding to NK cells.

[00143] In some embodiments, the polyspecific proteins described herein may provide a better safety profile by reducing non-tumor mistargeting. Natural killer cells and CD8 T cells are able to directly lyse tumor cells, although the mechanisms by which NK cells and CD8 T cells distinguish their own normal cells from tumor cells differ. The activity of NK cells is regulated by the balance of signals from activating (NCR, NKG2D, CD16, etc.) and inhibitory (KIR, NKG2A, etc.) receptors. The balance of these activating and inhibitory signals allows NK cells to identify healthy self-cells from stressed, virus-infected, or transformed self-cells. This "built-in" self-tolerance mechanism will help protect normal healthy tissue from NK cell responses. To extend this principle, NK cell self-tolerance would allow TriNKET to target both self-expressed antigens and tumor-expressed antigens, without the side effects of mistargeting or an extended therapeutic window. Unlike natural killer cells, T cells require recognition of a specific peptide presented by MHC molecules for activation and effector functions. T cells have been the main target of immunotherapy and many strategies have been developed to redirect T cell responses against the tumor. Bispecific T cells, checkpoint inhibitors, and CAR T cells have been approved by the FDA but often suffer from dose-limiting toxicity. Bispecific T cells and CAR-T cells function around the TCR-MHC recognition system, using binding domains to target antigens on the surface of tumor cells and using engineered signaling domains to signal activation to the effector cell. While these therapies are effective in eliciting an antitumor immune response, they are often associated with cytokine release syndrome (CRS) and non-tumor mistargeting side effects. Polyspecific proteins are unique in this context as they will not "override" the natural activation and inhibition systems of NK cells. Instead, polyspecific proteins are designed to maintain balance and provide additional activation signals to NK cells while maintaining NK tolerance to self cells.

[00144] In some embodiments, the polyspecific proteins described herein may more effectively delay tumor progression than the corresponding CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 monoclonal antibodies that comprise the same antigen-binding domain. In some embodiments, the polyspecific proteins described herein may be more effective against cancer metastases than the corresponding CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 monoclonal antibodies that include the same antigen-binding domain.

III. Therapeutic Applications

[00145] The invention relates to methods for treating cancer using the polyspecific binding protein described herein and/or the pharmaceutical composition described herein. The methods can be used to treat a variety of cancers whose cells express CAIX, ANO1, mesothelin, TROP2, CEA, or Claudin-18.2 by administering to a patient in need thereof a therapeutically effective amount of the polyspecific binding protein described herein.

[00146] The therapeutic method can be characterized depending on the malignant neoplasm to be treated. Cancers to be treated using CAIX-targeted polyspecific binding proteins include, for example, renal cell carcinoma, breast cancer, glioblastoma, head and neck cancer, gastric cancer, bladder cancer, ovarian cancer, tumors of the esophagus, lung, colon , kidney, cervical and non-small cell lung carcinoma. Cancers treatable with mesothelin-targeted polyspecific binding proteins include, for example, mesothelioma, ovarian cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, cholangiocarcinoma, gastric cancer, uterine serous carcinoma, thymic carcinoma, and acute myeloid leukemia. . Cancers treatable with ANO1-targeted polyspecific binding proteins include, for example, esophageal squamous cell carcinoma (ESCC), gastrointestinal stromal tumor (GIST), head and neck squamous cell carcinoma (HNSCC), pancreatic cancer, breast cancer , prostate cancer and sarcoma. Cancers to be treated with TROP2-targeted polyspecific binding proteins include, for example, breast, lung, stomach, colorectal, pancreatic, prostate, cervical, head and neck cancer, nasopharyngeal carcinoma, and ovarian carcinoma. Cancers to be treated with CEA-targeted polyspecific binding proteins include, for example, gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer. Cancers to be treated using Claudin-18.2-targeted polyspecific binding proteins include, for example, esophageal cancer, non-small cell lung carcinoma, ovarian cancer, colon cancer, and several forms of bile duct carcinoma.

[00147] In some other embodiments, the cancer is brain cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, rectal cancer, kidney cancer, stomach cancer, testicular cancer, or uterine cancer. In yet other embodiments, the cancer is squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, glandular squamous cell carcinoma, anal cancer, anal cancer, rectal cancer, astrocytic tumor, bartholin gland carcinoma, basal cell carcinoma, liver cancer, bone cancer , bone marrow cancer, bronchial cancer, bronchial gland carcinoma, carcinoid, cholangiocarcinoma, chondosarcoma, choriodapilloma/carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue cancer, cystadenoma, cancer of the digestive system, duodenal cancer, endocrine cancer endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cancer, ependymal cancer, epithelial cell carcinoma, Ewing's sarcoma, eye and orbital cancer, cancer of the female genital organs, focal nodular hyperplasia, gallbladder cancer, gastric cavity cancer, gastric fundus cancer, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangiblastoma, hemangioendothelioma, hemangiomas, hepatic adenoma, hepatic adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileum cancer, insulinoma, intraepithelial neoplasia, intraepithelial squamous neoplasia , intrahepatic bile duct cancer, invasive squamous cell carcinoma, small bowel cancer, connective tissue cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, lentigo melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothea lial tumors, medulloblastoma, medulloepithelioma, meningeal cancer, mesothelial cancer, metastatic cancer, oral cavity cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal canal cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer, osteosarcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, soft tissue cancer, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, striated muscle cancer, submesothelial cancer, superficial melanoma, T-cell leukemia, tongue cancer, undifferentiated cancer, cancer of the ureter, cancer of the urethra canal, bladder cancer, genitourinary tract cancer, cervical cancer, corpus uterine cancer, uveal melanoma, vaginal cancer, verrucous cancer, Vipoma, vulvar cancer, well-differentiated carcinoma, or Wilms tumor.

[00148] In some other embodiments, the cancer is a non-Hodgkin's lymphoma, such as B-cell lymphoma or T-cell lymphoma. In some embodiments, non-Hodgkin's lymphoma is a B-cell lymphoma, such as diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, B-cell extranodal marginal zone lymphoma, nodular marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary central nervous system (CNS) lymphoma. In some other embodiments, the non-Hodgkin's lymphoma is a T-cell lymphoma, such as progenitor T-cell lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural killer/T-cell lymphoma. nodular T-cell lymphoma, cutaneous T-cell lymphoma, enteropathic-type T-cell lymphoma, subcutaneous panniculitis-type T-cell lymphoma, anaplastic large cell lymphoma, or nodular T-cell lymphoma.

[00149] The cancer to be treated can be characterized according to the presence of a specific antigen expressed on the surface of the tumor cell. In some embodiments, the tumor cell may express one or more of the following: CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR/ERBB1, IGF1R, HER3/ERBB3, HER4/ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD1.

IV. Combination Therapy

[00150] Another aspect of the invention provides for combination therapy. The polyspecific binding proteins described herein are used in combination with additional therapeutic agents for the treatment of cancer.

[00151] Exemplary therapeutic agents that may be used as part of combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronit, methotrexate, doxorubicin, carboquone, pentostatin , nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxan, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxypfluridine, isotretinate, isotretinate, nimustine, vindesins, flutamide, drogenil, butocine, carmofur, razoxane, sizophilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocytabin, lisuride, oxymethetolone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colo non-stimulating factor-1, colony-stimulating factor-2, denileukin diphytox, interleukin-2, luteinizing hormone releasing factor, and variations of the aforementioned agents that may exhibit differential binding to their cognate receptor as well as increase or decrease serum half-life.

[00152] An additional class of agents that may be used as part of combination therapy in the treatment of cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, ( iv) LAG-3, (v) B7-H3, (vi) B7-H4 and (vii) TIM3. The CTLA4 inhibitor ipilimumab has been approved by the US Food and Drug Administration for the treatment of melanoma.

[00153] Other agents that may be used as part of combination therapy in the treatment of cancer are monoclonal antibody agents that target targets other than checkpoints (eg, Herceptin) and non-cytotoxic agents (eg, tyrosine kinase inhibitors) .

[00154] Other categories of antitumor agents include, for example: (i) an inhibitor selected from an ALK inhibitor, an ATR inhibitor, an A2A antagonist, a base exclusion repair inhibitor, a Bcr-Abl tyrosine kinase inhibitor, a Bruton tyrosine kinase inhibitor, a CDC7 inhibitor, a CHK1 inhibitor, a cyclin inhibitor α-dependent kinase, DNA-PK inhibitor, both DNA-PK and mTOR inhibitor, DNMT1 inhibitor, DNMT1 plus 2-chloro-deoxyadenosine inhibitor, HDAC inhibitor, Hedgehog signaling pathway inhibitor, IDO inhibitor, JAK inhibitor, mTOR inhibitor, MEK inhibitor , a MELK inhibitor, an MTH1 inhibitor, a PARP inhibitor, a phosphoinositide 3-kinase inhibitor, an inhibitor of both PARP1 and DHODH, a proteasome inhibitor, a topoisomerase-II inhibitor, a tyrosine kinase inhibitor, a VEGFR inhibitor, and a WEE1 inhibitor; (ii) an OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS agonist; and (iii) a cytokine selected from IL-12, IL-15, GM-CSF and G-CSF.

[00155] The proteins of the invention can also be used as an adjunct to surgical removal of the primary lesion.

[00156] The amount of polyspecific binding protein and additional therapeutic agent and the relative timing of administration can be selected to achieve the desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents in combination or the pharmaceutical composition or compositions containing the therapeutic agents may be administered in any order such as, for example, sequentially, simultaneously, together, simultaneously, and the like. In addition, for example, the multispecific binding protein may be administered during the time that the additional therapeutic agent(s) exerts its prophylactic or therapeutic effect, or vice versa.

V. Pharmaceutical compositions

[00157] The present disclosure also relates to pharmaceutical compositions that contain a therapeutically effective amount of the protein described herein. The composition can be formulated for use in various drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for a suitable composition. Suitable formulations for use in the present disclosure are Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed., 1985. For a brief review of drug delivery methods, see, for example, Langer (Science 249: 1527-1533, 1990) .

[00158] The intravenous drug delivery composition of the present invention may be contained in a bag, pen, or syringe. In some embodiments, the package may be connected to a channel containing a tube and/or a needle. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and may be contained in about 12-60 vials. In some embodiments, the composition may be lyophilized and 45 mg of the lyophilized composition may be contained in one vial. In some embodiments, about 40-100 mg of the lyophilized composition may be contained in one vial. In some embodiments, the lyophilized formulation of 12, 27, or 45 vials is combined to provide a therapeutic dose of protein in an intravenous formulation. In some embodiments, the composition may be a liquid composition and stored at a concentration of from about 250 mg/vial to about 1000 mg/vial. In certain embodiments, the composition may be a liquid composition and stored at a concentration of about 600 mg/vial. In some embodiments, the implementation of the composition may be a liquid composition and stored at a concentration of about 250 mg/vial.

[00159] The present disclosure may exist in a liquid aqueous pharmaceutical composition comprising a therapeutically effective amount of protein in a buffer solution to form a composition.

[00160] These compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle prior to administration. The pH of the formulations is typically 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, eg 7 to 7.5. The resulting solid form compositions may be packaged in multiple single dose units each containing a fixed amount of the above agent or agents. The solid form composition may also be packaged in a container for a flexible amount.

[00161] In some embodiments, the present disclosure relates to an extended shelf life formulation comprising a protein of the present invention in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[00162] In some embodiments, an aqueous formulation is prepared comprising a protein of the present invention in a pH buffer solution. The buffer of this invention may have a pH in the range of about 4 to about 8, such as about 4.5 to about 6, or about 4.8 to about 5.5, or may have a pH of about 5 to about 5.2. Ranges intermediate to the above pH values should also be part of this disclosure. For example, ranges of values using a combination of any of the above values as upper and/or lower limits are intended to be included. Examples of buffers that will control the pH within this range include acetate (eg sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

[00163] In certain embodiments, the composition includes a buffer system that contains citrate and phosphate to maintain a pH in the range of about 4 to about 8. In some embodiments, the pH range can be from about 4.5 to about 6, or about pH 4.8 to about 5.5, or in the pH range from about 5 to about 5.2. In some embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In some embodiments, the buffer system includes about 1.3 mg/mL citric acid (e.g., 1.305 mg/mL), about 0.3 mg/mL sodium citrate (e.g., 0.305 mg/mL), about 1.5 mg /ml disodium phosphate. dihydrate (eg 1.53 mg/ml), about 0.9 mg/ml sodium dihydrogen phosphate dihydrate (eg 0.86) and about 6.2 mg/ml sodium chloride (eg 6.165 mg/ml). In some embodiments, the buffer system comprises 1 to 1.5 mg/mL citric acid, 0.25 to 0.5 mg/mL sodium citrate, 1.25 to 1.75 mg/mL disodium phosphate dihydrate, 0, 7 to 1.1 mg/ml sodium dihydrogen phosphate dihydrate, and 6 to 6.4 mg/ml sodium chloride. In some embodiments, the pH of the composition is adjusted with sodium hydroxide.

[00164] A polyol that acts as a tonic and can stabilize an antibody can also be included. The polyol is added to the composition in an amount which may vary depending on the desired isotonicity of the composition. In some embodiments, the aqueous composition may be isotonic. The amount of added polyol can also be changed in relation to the molecular weight of the polyol. For example, a smaller amount of a monosaccharide (eg, mannitol) may be added compared to a disaccharide (such as trehalose). In some embodiments, the polyol that can be used as a tonicity agent in the formulation is mannitol. In certain embodiments, the concentration of mannitol may be from about 5 mg/mL to about 20 mg/mL. In some embodiments, the concentration of mannitol may be about 7.5-15 mg/mL. In some embodiments, the concentration of mannitol may be about 10-14 mg/ml. In certain embodiments, the concentration of mannitol may be about 12 mg/ml. In some embodiments, the implementation of the polyol sorbitol may be included in the composition.

[00165] A detergent or surfactant may also be added to the formulation. Typical detergents include non-ionic detergents such as polysorbates (eg polysorbates 20, 80, etc.) or poloxamers (eg poloxamer 188). The amount of detergent added is such that it reduces aggregation of the antibody in the formulation and/or minimizes particle formation in the formulation and/or reduces adsorption. In certain embodiments, the implementation of the composition may include a surfactant, which is a polysorbate. In some embodiments, the composition may contain detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In some embodiments, the composition may contain from about 0.1 to about 10 mg/ml of polysorbate 80, or from about 0.5 to about 5 mg/ml. In some embodiments, about 0.1% polysorbate 80 may be added to the composition.

[00166] In some embodiments, the implementation of the protein product of the present invention is formulated as a liquid composition. The liquid formulation may be presented at a concentration of 10 mg/mL in any USP/Ph Eur Type I 50R vial, rubber stoppered and sealed using an aluminum ferrule seal. The plug can be made from an elastomer that complies with USP and Ph Eur. In some embodiments, vials may be filled with 61.2 ml of protein product solution to provide an extractable volume of 60 ml. In some embodiments, the implementation of the liquid composition can be diluted with 0.9% saline.

[00167] In some embodiments, the implementation of the liquid composition according to the invention can be prepared in the form of a solution with a concentration of 10 mg/ml in combination with sugar at a stabilizing level. In some embodiments, the liquid composition may be formulated in an aqueous vehicle. In some embodiments, the implementation of the stabilizer may be added in an amount not exceeding that which may lead to a viscosity that is undesirable or unsuitable for intravenous administration. In some embodiments, the sugar may be disaccharides, such as sucrose. In some embodiments, the liquid composition may also include one or more of a buffering agent, a surfactant, and a preservative.

[00168] In some embodiments, the pH of the liquid formulation can be adjusted by adding a pharmaceutically acceptable acid and/or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the base may be sodium hydroxide.

[00169] In addition to aggregation, deamidation is a common product of peptides and proteins that can occur during fermentation, cell collection/clarification, purification, drug/drug product storage, and during sample analysis. Deamidation is the loss of NH3 from a protein to form an intermediate succinimide that can be hydrolysed. The intermediate succinimide results in a 17 dalton weight loss of the original peptide. Subsequent hydrolysis leads to an increase in mass by 18 daltons. Isolation of the intermediate succinimide is difficult due to instability in aqueous conditions. As such, deamidation is usually defined as an increase in mass by 1 dalton. Deamidation of asparagine results in aspartic or isoaspartic acid. Parameters affecting the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the rate of deamidation. Gly and Ser following Asn in protein sequences result in higher deamidation sensitivity.

[00170] In some embodiments, the implementation of the liquid composition of the present invention can be stored under conditions of pH and humidity to prevent deamination of the protein product.

[00171] The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for human administration) and useful in the preparation of a liquid composition. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00172] A preservative may optionally be added to formulations to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-dose (multi-dose) formulation.

[00173] Intravenous (IV) formulations may be the preferred route of administration in specific cases, for example, when the patient is in the hospital after transplant receiving all drugs via the IV route. In some embodiments, the liquid composition is diluted with 0.9% sodium chloride solution prior to administration. In some embodiments, the diluted injectable drug product is isotonic and suitable for administration by intravenous infusion.

[00174] In some embodiments, salts or buffer components may be added in an amount of 10-200 mM. The salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with base forming metals or amines. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case sodium, potassium, or ammonium ions may serve as the counterion.

[00175] A preservative may optionally be added to formulations to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-dose (multi-dose) formulation.

[00176] The aqueous carrier of interest herein is pharmaceutically acceptable (safe and non-toxic for human administration) and useful in the preparation of a liquid composition. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00177] The present disclosure may exist in a lyophilized composition comprising proteins and a lyoprotectant. The lyoprotectant may be sugar, such as disaccharides. In some embodiments, the lycoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a filler, and/or a preservative.

[00178] The amount of sucrose or maltose useful for stabilizing the lyophilized drug product may be in a weight ratio of at least 1:2 protein to sucrose or maltose. In some embodiments, the protein/sucrose or maltose weight ratio may be from 1:2 to 1:5.

[00179] In some embodiments, the pH of the composition prior to lyophilization can be adjusted by adding a pharmaceutically acceptable acid and/or base. In some embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In some embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

[00180] Prior to lyophilization, the pH of a solution containing a protein of the present invention may be adjusted in the range of 6 to 8. In some embodiments, the pH range of the lyophilized drug product may be 7 to 8.

[00181] In some embodiments, salts or buffer components may be added in an amount of 10-200 mM. The salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with base forming metals or amines. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case sodium, potassium, or ammonium ions may serve as the counterion.

[00182] In some embodiments, a "filler" may be added. A "filler" is a compound that adds bulk to the lyophilized mixture and contributes to the physical structure of the lyophilized tablet (eg, facilitates the production of a substantially uniform lyophilized tablet that maintains an open cell structure). Exemplary excipients include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such excipients.

[00183] A preservative may optionally be added to formulations to reduce bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-dose (multi-dose) formulation.

[00184] In some embodiments, the lyophilized drug product may consist of an aqueous carrier. The aqueous carrier of interest herein is pharmaceutically acceptable (eg, safe and non-toxic for human administration) and useful for preparing a liquid composition after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline, Ringer's solution, or dextrose solution.

[00185] In some embodiments, the lyophilized drug product of the present invention is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into the solution.

[00186] In some embodiments, the implementation of the lyophilized protein product of the present invention consists of about 4.5 ml of water for injection and is diluted with 0.9% saline (sodium chloride solution).

[00187] Actual dosage levels of active ingredients in pharmaceutical compositions may vary so as to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without toxicity to the patient.

[00188] The specific dose may be a unit dose for each patient, such as 50-5000 mg of protein. Alternatively, the dose to the patient may be adapted to the approximate body weight or surface area of the patient. Other factors in determining an appropriate dosage may include the disease or condition being treated or prevented, the severity of the disease, the route of administration, and the age, sex, and health of the patient. Those skilled in the art will typically further refine the calculations needed to determine an appropriate dosage for treatment, especially in light of the dosage and assay information disclosed herein. Dosage can also be determined by using known dose determination assays used in conjunction with appropriate dose-response data. The dosage of the individual patient may be adjusted as the progress of the disease is monitored. The blood levels of the targeting construct or complex in the patient can be measured to see if the dosage needs to be adjusted to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which targeting constructs and/or complexes and their doses are most likely to be effective in a given individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001).

[00189] Typically, doses based on body weight are from about 0.01 μg to about 100 mg per kg of body weight, for example, from about 0.01 μg to about 100 mg/kg of body weight, from about 0.01 µg to about 50 mg/kg body weight, about 0.01 µg to about 10 mg/kg body weight, about 0.01 µg to about 1 mg/kg body weight, about 0.01 µg to about 100 µg /kg of body weight, from about 0.01 µg to about 50 µg/kg of body weight, from about 0.01 µg to about 10 µg/kg of body weight, from about 0.01 µg to about 1 µg/kg of body weight, from about 0.01 µg to about 0.1 µg/kg body weight, from about 0.1 µg to about 100 mg/kg body weight, from about 0.1 µg to about 50 mg/kg body weight, from about 0 1 µg to about 10 mg/kg body weight, about 0.1 µg to about 1 mg/kg body weight, about 0.1 µg to about 100 µg/kg body weight, about 0.1 µg to about 50 µg/kg body weight, about 0.1 µg to about 10 µg/kg body weight, example but from 0.1 µg to about 1 µg/kg body weight, from about 1 µg to about 100 mg/kg body weight, from about 1 µg to about 50 mg/kg body weight, from about 1 µg to about 10 mg/kg kg body weight, from about 1 µg to about 1 mg/kg body weight, from about 1 µg to about 100 µg/kg body weight, from about 1 µg to about 50 µg/kg body weight, from about 1 µg to about 10 µg/kg body weight, from about 10 µg to about 100 mg/kg body weight, from about 10 µg to about 50 mg/kg body weight, from about 10 µg to about 10 mg/kg body weight, from about 10 µg from about up to 1 mg/kg body weight, from about 10 µg to about 100 µg/kg body weight, from about 10 µg to about 50 µg/kg body weight, from about 50 µg to about 100 mg/kg body weight, from about 50 µg to about 50 mg/kg body weight, about 50 µg to about 10 mg/kg body weight, about 50 µg to about 1 mg/kg body weight, about 50 µg to about 100 µg/kg body weight, about from 100 µg to about 100 mg/kg body weight, about 100 µg to about 50 mg/kg body weight, about 100 µg to about 10 mg/kg body weight, about 100 µg to about 1 mg/kg body weight, about 1 mg to about 100 mg/kg body weight, about 1 mg to about 50 mg/kg body weight, about 1 mg to about 10 mg/kg body weight, about 10 mg to about 100 mg/kg body weight , from about 10 mg to about 50 mg/kg of body weight, from about 50 mg to about 100 mg/kg of body weight.

[00190] Doses may be administered one or more times a day, weekly, monthly or yearly, or even once every 2-20 years. Those skilled in the art can easily estimate the repetition rate for dosing based on the measured retention time and concentrations of the targeting construct or complex in body fluids or tissues. The administration of the present invention may be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by catheter perfusion, or by direct intracellular injection. This can be done one or more times a day, one or more times a week, one or more times a month, and one or more times a year.

[00191] The above description describes many aspects and embodiments of the invention. The patent application specifically addresses all combinations and permutations of aspects and embodiments.

EXAMPLES

[00192] The present invention has now been generally described and will be better understood with reference to the following examples, which are included for the purpose of illustrating certain aspects and embodiments of the present invention only and are not intended to limit the invention in any way.

Example 1 NKG2D Binding Domains Bind to NKG2D

NKG2D binding domains bind to purified recombinant NKG2D

[00193] Nucleic acid sequences of human, mouse, or cynomolgus monkey NKG2D ectodomains were fused to nucleic acid sequences encoding human IgG1 Fc domains and introduced into mammalian cells for expression. After purification, NKG2D-Fc fusion proteins were adsorbed into microplate wells. After blocking the wells with bovine serum albumin to prevent non-specific binding, the NKG2D binding domains were titrated and added to the wells to which the NKG2D-Fc fusion proteins had previously been adsorbed. Primary antibody binding was determined using a secondary antibody that is conjugated to horseradish peroxidase and specifically recognizes the human kappa light chain to avoid Fc cross-reactivity. 3,3',5,5'-tetramethylbenzidine (TMB), a substrate for horseradish peroxidase, was added to the wells to visualize the binding signal, the absorbance of which was measured at 450 nM and corrected at 540 nM. An NKG2D binding domain clone, isotype control or positive control (selected from SEQ ID NOs: 47-50 or NKG2D anti-mouse clones MI-6 and CX-5 available from eBioscience) was added to each well.

[00194] The isotype control showed minimal binding to recombinant NKG2D-Fc proteins, while the positive control binds most strongly to recombinant antigens. The NKG2D binding domains produced by all clones exhibited binding to recombinant human, mouse, and cynomolgus monkey NKG2D-Fc proteins, although with varying affinities from clone to clone. Typically, each anti-NKG2D clone binds to recombinant human NKG2D-Fc (FIG. 3) and cynomolgus macaque (FIG. 4) with similar affinity but lower affinity to recombinant mouse NKG2D-Fc (FIG. 5).

NKG2D binding domains bind to cells expressing NKG2D

[00195] Murine EL4 lymphoma cell lines were engineered to express the chimeric NKG2D antigen receptor and human or mouse CD3-zeta signaling domain. An NKG2D binding clone, isotype control or positive control was used at 100 nM to stain for extracellular NKG2D expressed on EL4 cells. Antibody binding was determined using secondary anti-human IgG antibodies conjugated to a fluorophore. Cells were analyzed by flow cytometry and fold-over-background (FOB) was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[00196] NKG2D-binding domains produced by all clones associated with EL4 cells expressing human and mouse NKG2D. Positive control antibodies (selected from SEQ ID NOs: 45-48 or NKG2D anti-mouse clones MI-6 and CX-5 available from eBioscience) gave the best FOB binding signal. NKG2D binding affinity for each clone was similar between cells expressing NKG2D human (FIG. 6) and mouse (FIG. 7).

Example 2 NKG2D Binding Domains Block Natural Ligand Binding to NKG2D

Competition with ULBP-6

[00197] Recombinant human NKG2D-Fc proteins were adsorbed into microplate wells, and the wells were blocked with bovine serum albumin to reduce non-specific binding. A saturated concentration of ULBP-6-His-biotin was added to the wells followed by the addition of clones of the NKG2D binding domain. After a 2 hour incubation, the wells were washed and ULBP-6-His-biotin, which remained bound to the NKG2D-Fc coated wells, was detected with streptavidin conjugated to horseradish peroxidase and TMB substrate. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D binding domains to NKG2D-Fc proteins was calculated from the percentage of ULBP-6-His-biotin that was blocked from binding to NKG2D-Fc proteins in the wells. The positive control antibody (selected from SEQ ID NO: 47-50) and various NKG2D binding domains blocked ULBP-6 binding to NKG2D, while the isotype control showed little competition with ULBP-6 (FIG. 8).

[00198] The sequence of ULBP-6 is shown in SEQ ID NO: 131

MAAAAIPALLLCLPLLFLLFGWSRARRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKLNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARMSCEQKAEGHSSGSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSGTTQLRATATTLILCCLLIILPCFILPGI (SEQ ID NO: 131)

Competition with MICA

[00199] Recombinant human MICA-Fc proteins were adsorbed into microplate wells, and the wells were blocked with bovine serum albumin to reduce non-specific binding. NKG2D-Fc-biotin was added to the wells, followed by the addition of NKG2D-binding domains. After incubation and washing with NKG2D-Fc, biotin that remained bound to MICA-Fc coated wells was detected using streptavidin-HRP substrate and TMB. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D binding domains to NKG2D-Fc proteins was calculated from the percentage of NKG2D-Fc-biotin that was blocked from binding to MICA-Fc in the wells. The positive control antibody (selected from SEQ ID NO: 47-50) and various NKG2D binding domains blocked MICA binding to NKG2D, while the isotype control showed little competition with MICA (FIG. 9).

Competition with Rae-1 Delta

[00200] Recombinant mouse Rae-1delta-Fc (purchased from R&D Systems) was adsorbed into microplate wells and the wells were blocked with bovine serum albumin to reduce non-specific binding. Mouse NKG2D-Fc-biotin was added to the wells followed by the addition of NKG2D binding domains. After incubation and washing with NKG2D-Fc, biotin that remained bound to Rae-1delta-Fc coated wells was detected using streptavidin-HRP substrate and TMB. Absorbance was measured at 450 nM and corrected at 540 nM. After background subtraction, specific binding of NKG2D binding domains to NKG2D-Fc proteins was calculated from the percentage of NKG2D-Fc-biotin that was blocked from binding to Rae-1delta-Fc coated wells. The positive control (selected from SEQ ID NO: 47-50 or NKG2D anti-mouse clones MI-6 and CX-5 available from eBioscience) and various NKG2D-binding domain clones blocked Rae-1-delta binding to mouse NKG2D, while while the isotype control antibody showed little competition with Rae-1delta (FIG. 10).

Example 3 NKG2D Binding Domain Clones Activate NKG2D

[00201] Human and mouse NKG2D nucleic acid sequences were fused to nucleic acid sequences encoding the CD3 zeta signaling domain to generate chimeric antigen receptor (CAR) constructs. The NKG2D-CAR constructs were then cloned into a retroviral vector using a Gibson assembly and transfected into expi293 cells to generate a retrovirus. EL4 cells were infected with viruses containing NKG2D-CAR along with 8 μg/ml polybrene. 24 hours after infection, NKG2D-CAR expression levels in EL4 cells were analyzed by flow cytometry and clones were selected that highly express NKG2D-CAR on the cell surface.

[00202] To determine if the NKG2D binding domains of NKG2D are activated, they are adsorbed into microplate wells and NKG2D-CAR EL4 cells are cultured in antibody fragment coated wells for 4 hours in the presence of brefeldin-A and monensin. Intracellular production of TNF-alpha, an indicator of NKG2D activation, was analyzed by flow cytometry. The percentage of TNF-alpha positive cells was normalized to cells treated with a positive control. All NKG2D binding domains activated both human (FIG. 11) and mouse (FIG. 12) NKG2D.

Example 4 NKG2D Binding Domains Activate NK Cells

Primary human NK cells

[00203] Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood buffy coat using density gradient centrifugation. NK cells (CD3-CD56+) were isolated using negative screening with PBMC magnetic beads, and the purity of the isolated NK cells was typically >95%. Then, the isolated NK cells were cultured in a medium containing 100 ng/ml IL-2 for 24-48 hours, after which they were transferred to the wells of a microplate on which NKG2D-binding domains were adsorbed, and cultured in a medium containing conjugated with fluorophore anti-CD107a antibody, brefeldin-A and monensin. After culture, NK cells were analyzed by flow cytometry using fluorophore-conjugated antibodies against CD3, CD56 and IFN-γ. CD107a and IFN-γ staining was analyzed in CD3-CD56+ cells to assess NK cell activation. An increase in CD107a/IFN-γ double positive cells indicates better NK cell activation by recruiting two activating receptors rather than one receptor. NKG2D binding domains and a positive control (selected from SEQ ID NOs: 45-48) showed a higher percentage of NK cells becoming CD107a+ and IFN-γ+ than the isotype control (FIG. 13 and FIG. 14 represent two independent experiments, each of which used different PBMC donors to prepare NK cells).

Primary mouse NK cells

[00204] Spleens were obtained from C57Bl/6 mice and ground through a 70 µm cell sieve to obtain a single cell suspension. Cells were pelleted and resuspended in ACK lysis buffer (purchased from Thermo Fisher Scientific # A1049201; 155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mMEDTA) to remove erythrocytes. The remaining cells were cultured with 100 ng/ml hIL-2 for 72 hours and then harvested and prepared for NK cell isolation. NK cells (CD3-NK1.1+) were then isolated from spleen cells using a negative depletion method using magnetic beads with >90% purity. Purified NK cells were cultured in a medium containing 100 ng/ml mIL-15 for 48 hours, after which they were transferred to the wells of a microplate on which NKG2D-binding domains were adsorbed and cultured in a medium containing a fluorophore-conjugated antibody against CD107a, brefeldin-A and monensin. After culture in wells coated with the NKG2D binding domain, NK cells were analyzed by flow cytometry using fluorophore-conjugated antibodies against CD3, NK1.1 and IFN-γ. CD107a and IFN-γ staining was analyzed in CD3-NK1.1+ cells to assess NK cell activation. An increase in the number of CD107a/IFN-γ double positive cells indicates better activation of NK cells due to the involvement of two activating receptors rather than one receptor. NKG2D binding domains and a positive control (selected from anti-mouse NKG2D clones MI-6 and CX-5 available from eBioscience) showed a higher percentage of NK cells becoming CD107a+ and IFN-γ+ than the isotype control (FIG. 15 and FIG 16 represent two independent experiments, each using different mice to prepare NK cells).

Example 5 NKG2D Binding Domains Confer Cytotoxicity in Target Tumor Cells

[00205] Primary human and mouse NK cell activation assays demonstrate increased levels of cytotoxicity markers for NK cells after incubation with NKG2D binding domains. To determine if this results in increased tumor cell lysis, a cellular assay was used in which each NKG2D binding domain was converted into a monospecific antibody. The Fc region was used as one targeting fragment, while the Fab region (NKG2D binding domain) acted as the other targeting fragment to activate NK cells. THP-1 cells, which are of human origin and highly express Fc receptors, were used as a tumor target and the Perkin Elmer DELFIA cytotoxicity kit was used. THP-1 cells were labeled with BATDA reagent and resuspended at 105/ml in culture medium. Labeled THP-1 cells were then combined with NKG2D antibodies and isolated mouse NK cells in microplate wells at 37° C. for 3 hours. After incubation, 20 μl of culture supernatant was removed, mixed with 200 μl of europium solution and incubated with shaking for 15 minutes in the dark. Fluorescence was measured over time using a PheraStar plate reader equipped with a time-resolved fluorescence module (337 nm excitation, 620 nm emission) and specific lysis was calculated according to the kit instructions.

[00206] The positive control, ULBP-6, a natural ligand for NKG2D, showed increased specific lysis of THP-1 target cells by mouse NK cells. NKG2D antibodies also increased the specific lysis of THP-1 target cells, while the isotype control antibody showed a decrease in specific lysis. The dotted line indicates specific lysis of THP-1 cells by mouse NK cells without the addition of antibodies (FIG. 17).

Example 6 NKG2D Antibodies Show High Thermal Stability

[00207] The melting point of NKG2D binding domains was analyzed using differential scanning fluorometry. Extrapolated apparent melting points are high compared to typical IgG1 antibodies (FIG. 18).

Example 7 Synergistic activation of human NK cells by cross-linking NKG2D and CD16.

Primary human NK cell activation assay

[00208] Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood buffy coat using density gradient centrifugation. NK cells were purified from PBMC using negative magnetic beads (StemCell # 17955). NK cells were >90% CD3-CD56+ as determined by flow cytometry. Cells were then grown 48 hours in medium containing 100 ng/ml hIL-2 (Peprotech # 200-02) before being used in activation assays. Antibodies were applied to a 96-well flat bottom plate at 2 µg/ml (anti-CD16, Biolegend # 302013) and 5 µg/ml (anti-NKG2D, R&D # MAB139) in 100 µl sterile PBS overnight at 4°C followed by thorough washing of the wells to remove excess antibodies. To evaluate degranulation, IL-2-activated NK cells were resuspended at 5 x 105 cells/ml in culture medium supplemented with 100 ng/ml hIL2 and 1 μg/ml APC-conjugated anti-CD107a mAb (Biolegend # 328619). Then 1 x 105 cells/well were added to the antibody coated plates. The protein transport inhibitors Brefeldin A (BFA, Biolegend # 420601) and Monensin (Biolegend # 420701) were added at a final dilution of 1:1000 and 1:270, respectively. Seeded cells were incubated for 4 hours at 37°C in 5% CO2. For intracellular IFN-γ staining, NK cells were labeled with anti-CD3 (Biolegend # 300452) and anti-CD56 mAb (Biolegend # 318328) and then fixed and permeabilized and labeled with anti-IFN-γ mAb (Biolegend # 506507). NK cells were analyzed for CD107a and IFN-γ expression by flow cytometry after gating live CD56+CD3 cells.

[00209] To investigate the relative potency of the receptor combination, NKG2D or CD16 cross-linking and co-binding of both receptors via stimulation in the tablet-bound state was performed. As shown in Figure. 19 (FIGS. 19A-19C), combined stimulation of CD16 and NKG2D resulted in greatly elevated CD107a levels (degranulation) (FIG. 19A) and/or IFN-γ production (FIG. 19B). The dotted lines represent the additive effect of individual stimulations of each receptor.

[00210] CD107a levels and intracellular IFN-γ production of activated IL-2 NK cells were analyzed after 4 hours of plate-bound stimulation with anti-CD16, anti-NKG2D, or a combination of both monoclonal antibodies. Plots show mean (n=2) ± SD. FIG. 19A shows the level of CD107a; FIG. 19B shows the level of IFNγ; FIG.19C shows the level of CD107a and IFNγ. The data shown in FIG. 19A-19C represent five independent experiments using five different healthy donors.

Example 8 Multispecific Binding Proteins Bind to NKG2D

[00211] Mouse EL4 lymphoma cell lines were engineered to express human NKG2D. Trispecific binding proteins (TriNKET), each containing a NKG2D binding domain, a tumor specific antigen binding domain (CAIX binding domain), and an Fc domain that binds to CD16, as shown in FIG. 1 were tested for their affinity for extracellular NKG2D expressed on EL4 cells. Binding of multispecific binding proteins to NKG2D was determined using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and fold-over-background (FOB) was calculated using the mean fluorescence intensity (MFI) of NKG2D-expressing cells compared to parental EL4 cells.

[00212] TriNKETs tested include A44-TriNKET-CAIX (ADI-27744 and CAIX binding domain), F63-TriNKET-CAIX (ADI-29404 and CD33 binding domain), E79-TriNKET-CAIX (ADI-29379 and CAIX- binding domain), F47-TriNKET-CAIX (ADI-29447 and CAIX binding domain) and A49-TriNKET-CAIX (ADI-27749 and CAIX binding domain). The CAIX binding domain consisted of the heavy chain variable domain of SEQ ID NO: 80 and the light chain variable domain of SEQ ID NO: 81.

[00213] FIG. 35 shows that TriNKETs, each including a CAIX binding domain and a different NKG2D binding domain, bind to NKG2D expressed on EL4 cells.

Example 9 Polyspecific Binding Proteins Bind to Human Tumor Antigens

Trispecific binding proteins bind to CAIX

[00214] The A498 human renal cell carcinoma line expressing CAIX was used to analyze the binding of TriNKET to the CAIX tumor-specific antigen. TriNKET and optionally the parent anti-CAIX monoclonal antibody were incubated with the cells and binding was detected using fluorophore-conjugated anti-human IgG secondary antibodies. Cells were analyzed by flow cytometry and excess over background (FOB) was calculated using mean fluorescence intensity (MFI) from TriNKET and parental anti-CAIX monoclonal antibody normalized to secondary antibody controls. TriNKETs targeting CAIX show comparable levels of binding to CAIX on A498 cells compared to the parental anti-CAIX antibody (FIG. 36). The overall binding signal is comparable among TriNKETs as they contain the same CAIX binding domain.

Example 10 Trispecific binding proteins provide cytotoxicity to tumor target cells.

[00215] Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood buffy coat using density gradient centrifugation. NK cells (CD3-CD56+) were isolated using negative screening with PBMC magnetic beads, and the purity of the isolated NK cells was typically >90%. The isolated NK cells were cultured in a medium containing 100 ng/ml IL-2 for activation or left overnight without the cytokine. IL-2-activated or resting NK cells were used the next day in cytotoxicity assays.

[00216] NK/NK T cell lines KHYG-1 were maintained in 10% HI-FBS-RPMI-1640 with 10 ng/ml IL-2. The day before use as effector cells, KHYG-1 cells were harvested from culture and the cells were washed from medium containing IL-2. After washing, KHYG-1 cells were resuspended in 10% HI-FBS-RPMI-1640 and left overnight without cytokine.

DELFIA cytotoxicity assay:

[00217] Human A498 tumor cells expressing CAIX were harvested from culture; cells were washed with PBS and resuspended in growth medium at 106/ml for labeling with BATDA reagent (Perkin Elmer AD0116). The manufacturer's instructions for labeling target cells were followed. After labeling, the cells were washed 3 times with PBS and resuspended in the amount of 0.5-1.0 x 105/ml in the culture medium. To prepare background wells, an aliquot of labeled cells was taken, and the cells were removed from the medium. 100 µl of medium was carefully added to the wells in triplicate so as not to damage the precipitated cells. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. The wells were protected for spontaneous release from target cells and the wells were prepared for maximum target cell lysis by adding 1% Triton-X. TriNKET against the target tumor of interest was diluted in culture medium, 50 μl of diluted TriNKET was added to each well. Resting NK cells, activated NK cells or KHYG-1 cells were collected from the culture, washed and resuspended at 105-2.0 x 106/ml in culture medium depending on the desired ratio of effector to target (E:T). 50 μl of NK cells were added to each well of the plate to give a total of 200 μl of culture volume. The plate was incubated at 37°C with 5% CO2 for 2-3 hours before analysis.

[00218] After culturing for 2-3 hours, the plate was removed from the incubator and the cells were pelleted by centrifugation at 200g for 5 minutes. 20 µl of the culture supernatant was transferred to a clean microplate provided by the manufacturer, and 200 µl of europium solution at room temperature was added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes. The plate was read using Victor 3 or SpectraMax i3X instruments. The % specific lysis was calculated as follows: % specific lysis = ((Experimental release - spontaneous release)/(Maximum release - spontaneous release)) * 100%.

[00219] TriNKETs mediate the cytotoxicity of human NK cells to CAIX-positive human tumor cells. Co-cultivation of resting KHYG-1 cells with A498 target cells resulted in ~38% specific tumor lysis (shown by dotted line in FIG. 37A). Addition of three different CAIX-targeting TriNKETs (A49-TriNKET-CAIX; E79-TriNKET-CAIX; and A44-TriNKET-CAIX) to co-culture enhanced specific tumor cell lysis by approximately 20% for all three NKG2D-binding domains (FIG 37A).

[00220] Resting human NK cells were mixed with A498 tumor cells. Resting human NK cells had no cytotoxic activity against A498 cells without the addition of CAIX mAb or TriNKET. An anti-CAIX monoclonal antibody (BAY79-4620 variable domains with hIgG1 as mAb (homodimer) was able to increase specific lysis by about 9%. cytotoxic activity of resting human NK cells against tumor cells compared to monoclonal antibody (FIG. 37B).

ENABLE USING THE LINK

[00221] The full disclosure of each of the patent documents and scientific articles referenced herein is incorporated by reference for all purposes.

EQUIVALENTS

[00222] The invention may be embodied in other specific forms without deviating from its spirit or essential characteristics. Thus, the above embodiments should be considered in all respects as illustrative and not limiting of the invention described herein. Thus, the scope of the invention is indicated by the appended claims and not by the preceding description, and all changes that fall within the meaning and range of equivalence of the claims are intended to be included therein.

--->

SEQUENCE LIST

<110> ADIMAB, LLC.

<120> POLYSPECIFIC BINDING PROTEINS TARGETING CAIX, ANO1,

MESOTELIN, TROP2, CEA OR CLAUDIN-18.2

<130> DFY-012WO

<140>

<141>

<150> 62/473.659

<151> 2017-03-20

<150> 62/473.652

<151> 2017-03-20

<150> 62/467.557

<151> 2017-03-06

<150> 62/464.347

<151> 2017-02-27

<150> 62/464.344

<151> 2017-02-27

<150> 62/464.341

<151> 2017-02-27

<160> 236

<170>PatentIn version 3.5

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Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 2

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Ile

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 3

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 3

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 4

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

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<400> 4

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

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Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

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Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 5

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

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<400> 5

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 6

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 6

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 7

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 7

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 8

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 8

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Ser Tyr Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 9

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 10

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 10

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 11

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 11

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Gly Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 12

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 12

Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Ile Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 13

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 14

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Ile

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 15

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 16

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Glu Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 17

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 17

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 18

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 18

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 19

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 19

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 20

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 20

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ile Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 21

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 21

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 22

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 23

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 24

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gly Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 25

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 26

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 27

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 28

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Phe Ser Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 29

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 30

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Ser Tyr Ser Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 31

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 31

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 32

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 32

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Phe Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 33

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 33

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 34

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Gln Ser Tyr Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 35

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 35

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 36

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 36

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 37

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 37

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 38

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 38

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Glu Leu Tyr Ser Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 39

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 39

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 40

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 40

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Thr Phe Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 41

<211> 125

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Gly Met

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 42

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 42

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Thr Pro Ile Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 43

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 43

Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser

20 25 30

Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 44

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 44

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Asp Thr Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 45

<211> 126

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Gly Arg Lys Ala Ser Gly Ser Phe Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 46

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 46

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Glu Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Pro Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 47

<211> 121

<212> Protein

<213> Homo sapiens

<400> 47

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Arg Gly Leu Gly Asp Gly Thr Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 48

<211> 110

<212> Protein

<213> Homo sapiens

<400> 48

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Phe Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 49

<211> 115

<212> Protein

<213> Homo sapiens

<400> 49

Gln Val His Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Asp Asp Ser Ile Ser Ser Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly His Ile Ser Tyr Ser Gly Ser Ala Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Asn Trp Asp Asp Ala Phe Asn Ile Trp Gly Gln Gly Thr Met Val Thr

100 105 110

Val Ser Ser

115

<210> 50

<211> 108

<212> Protein

<213> Homo sapiens

<400> 50

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 51

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 51

Gly Ser Phe Ser Gly Tyr Tyr Trp Ser

15

<210> 52

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 52

Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 53

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 53

Ala Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro

1 5 10

<210> 54

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 54

Gly Thr Phe Ser Ser Tyr Ala Ile Ser

15

<210> 55

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 55

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 56

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 56

Ala Arg Gly Asp Ser Ser Ile Arg His Ala Tyr Tyr Tyr Tyr Gly Met

1 5 10 15

Asp Val

<210> 57

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 57

Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 58

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 58

Trp Ala Ser Thr Arg Glu Ser

15

<210> 59

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 59

Gln Gln Tyr Tyr Ser Thr Pro Ile Thr

15

<210> 60

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 60

Gly Ser Ile Ser Ser Ser Ser Tyr Tyr Trp Gly

1 5 10

<210> 61

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 61

Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 62

<211> 13

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 62

Ala Arg Gly Ser Asp Arg Phe His Pro Tyr Phe Asp Tyr

1 5 10

<210> 63

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 63

Arg Ala Ser Gln Ser Val Ser Arg Tyr Leu Ala

1 5 10

<210> 64

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 64

Asp Ala Ser Asn Arg Ala Thr

15

<210> 65

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 65

Gln Gln Phe Asp Thr Trp Pro Pro Thr

15

<210> 66

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 66

Gly Thr Phe Ser Ser Tyr Ala Ile Ser

15

<210> 67

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 67

Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 68

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 68

Ala Arg Arg Gly Arg Lys Ala Ser Gly Ser Phe Tyr Tyr Tyr Tyr Gly

1 5 10 15

Met Asp Val

<210> 69

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 69

Glu Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 70

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 70

Trp Ala Ser Thr Arg Glu Ser

15

<210> 71

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 71

Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr

15

<210> 72

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 72

Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val

35 40 45

Ala Ala Ile Asn Ser Asp Gly Gly Ile Thr Tyr Tyr Leu Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Phe Tyr Cys

85 90 95

Ala Arg His Arg Ser Gly Tyr Phe Ser Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 73

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 73

Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Thr Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Val Ser Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Phe Phe Cys Gln Gln Tyr Ser Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 74

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 74

Gly Phe Thr Phe Ser Asn Tyr

15

<210> 75

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 75

Asn Ser Asp Gly Gly Ile

15

<210> 76

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 76

His Arg Ser Gly Tyr Phe Ser Met Asp Tyr

1 5 10

<210> 77

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 77

Gln Asn Val Val Ser Ala Val Ala

15

<210> 78

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 78

Ser Ala Ser Asn Arg Tyr Thr

15

<210> 79

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 79

Gln Gln Tyr Ser Asn Tyr Pro Trp Thr

15

<210> 80

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 80

Gln Val Glu Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Ser Ser Leu Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Ser Pro Gly Thr Phe Met His Gly Asp His Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 81

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 81

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Gly Arg Pro Thr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 82

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 82

Gly Phe Thr Phe Ser Tyr Gly Met Ser

1 5 10

<210> 83

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 83

Gly Ile Ser Ser Leu Gly Ser Thr Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 84

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 84

Thr Gly Ser Pro Gly Thr Phe Met His Gly Asp His

1 5 10

<210> 85

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 85

Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Ser

1 5 10

<210> 86

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 86

Tyr Gly Ala Ser Asn Leu Gln Ser

15

<210> 87

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 87

Gln Gln Tyr Tyr Gly Arg Pro Thr

15

<210> 88

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 88

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Leu Val

35 40 45

Ser Ala Ile Asn Ser Asp Gly Gly Ile Thr Tyr Tyr Leu Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Gly Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Arg Ser Gly Tyr Phe Ser Met Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 89

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 89

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Val Val Ser Ala

20 25 30

Val Ala Trp Tyr Leu Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Asn Tyr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 90

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 90

Gly Phe Thr Phe Ser Asn Tyr

15

<210> 91

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 91

Asn Ser Asp Gly Gly Ile

15

<210> 92

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 92

His Arg Ser Gly Tyr Phe Ser Met Asp Tyr

1 5 10

<210> 93

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 93

Gln Asn Val Val Ser Ala Val Ala

15

<210> 94

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 94

Ser Ala Ser Asn Arg Tyr Thr

15

<210> 95

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 95

Gln Gln Tyr Ser Asn Tyr Pro Trp Thr

15

<210> 96

<211> 459

<212> Protein

<213> Unknown

<220>

<223> Description of unknown:

CAIX polypeptide

<400> 96

Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu Ile Pro Ala

1 5 10 15

Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu Leu Leu Leu

20 25 30

Val Pro Val His Pro Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro

35 40 45

Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp

50 55 60

Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu

65 70 75 80

Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro

85 90 95

Glu Val Lys Pro Lys Ser Glu Glu Glu Gly Ser Leu Lys Leu Glu Asp

100 105 110

Leu Pro Thr Val Glu Ala Pro Gly Asp Pro Gln Glu Pro Gln Asn Asn

115 120 125

Ala His Arg Asp Lys Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly

130 135 140

Gly Asp Pro Pro Trp Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe

145 150 155 160

Gln Ser Pro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala

165 170 175

Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu

180 185 190

Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro

195 200 205

Gly Leu Glu Met Ala Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu Gln

210 215 220

Leu His Leu His Trp Gly Ala Ala Gly Arg Pro Gly Ser Glu His Thr

225 230 235 240

Val Glu Gly His Arg Phe Pro Ala Glu Ile His Val Val His Leu Ser

245 250 255

Thr Ala Phe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu

260 265 270

Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala

275 280 285

Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser

290 295 300

Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp

305 310 315 320

Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr Pro Pro Cys

325 330 335

Ala Gln Gly Val Ile Trp Thr Val Phe Asn Gln Thr Val Met Leu Ser

340 345 350

Ala Lys Gln Leu His Thr Leu Ser Asp Thr Leu Trp Gly Pro Gly Asp

355 360 365

Ser Arg Leu Gln Leu Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg

370 375 380

Val Ile Glu Ala Ser Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala

385 390 395 400

Ala Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu

405 410 415

Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu

420 425 430

Val Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys Gly Gly Val Ser

435 440 445

Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Ala

450 455

<210> 97

<211> 123

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 97

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr

20 25 30

Asp Met His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Asp Trp Met

35 40 45

Gly Val Ile Trp Gly Asn Gly Lys Thr Gln Tyr Asn Ser Gly Leu Thr

50 55 60

Ser Arg Leu Ser Ile Ser Arg Asp Thr Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Ile Tyr Phe Cys Thr

85 90 95

Arg Ser Gly Tyr Tyr Tyr Asp Gly Ser Tyr Tyr Ser Leu Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Val Met Val Thr Val Ser Ser

115 120

<210> 98

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 98

Asp Val Val Leu Thr Gln Thr Pro Pro Thr Leu Ser Ala Thr Ile Gly

1 5 10 15

Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Pro

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Val Ser Arg Leu Glu Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Ser

85 90 95

Thr His Ala Pro Ala Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 99

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 99

Asn Tyr Asp Met His

15

<210> 100

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 100

Val Ile Trp Gly Asn Gly Lys Thr Gln Tyr Asn Ser Gly Leu Thr Ser

1 5 10 15

<210> 101

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 101

Ser Gly Tyr Tyr Tyr Asp Gly Ser Tyr Tyr Ser Leu Phe Asp Tyr

1 5 10 15

<210> 102

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 102

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> 103

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 103

Leu Val Ser Arg Leu Glu Ser

15

<210> 104

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 104

Val Gln Ser Thr His Ala Pro Ala

15

<210> 105

<211> 986

<212> Protein

<213> Unknown

<220>

<223> Description of unknown:

ANO1 polypeptide

<400> 105

Met Arg Val Asn Glu Lys Tyr Ser Thr Leu Pro Ala Glu Asp Arg Ser

1 5 10 15

Val His Ile Ile Asn Ile Cys Ala Ile Glu Asp Ile Gly Tyr Leu Pro

20 25 30

Ser Glu Gly Thr Leu Leu Asn Ser Leu Ser Val Asp Pro Asp Ala Glu

35 40 45

Cys Lys Tyr Gly Leu Tyr Phe Arg Asp Gly Arg Arg Lys Val Asp Tyr

50 55 60

Ile Leu Val Tyr His His Lys Arg Pro Ser Gly Asn Arg Thr Leu Val

65 70 75 80

Arg Arg Val Gln His Ser Asp Thr Pro Ser Gly Ala Arg Ser Val Lys

85 90 95

Gln Asp His Pro Leu Pro Gly Lys Gly Ala Ser Leu Asp Ala Gly Ser

100 105 110

Gly Glu Pro Pro Met Asp Tyr His Glu Asp Asp Lys Arg Phe Arg Arg

115 120 125

Glu Glu Tyr Glu Gly Asn Leu Leu Glu Ala Gly Leu Glu Leu Glu Arg

130 135 140

Asp Glu Asp Thr Lys Ile His Gly Val Gly Phe Val Lys Ile His Ala

145 150 155 160

Pro Trp Asn Val Leu Cys Arg Glu Ala Glu Phe Leu Lys Leu Lys Met

165 170 175

Pro Thr Lys Lys Met Tyr His Ile Asn Glu Thr Arg Gly Leu Leu Lys

180 185 190

Lys Ile Asn Ser Val Leu Gln Lys Ile Thr Asp Pro Ile Gln Pro Lys

195 200 205

Val Ala Glu His Arg Pro Gln Thr Met Lys Arg Leu Ser Tyr Pro Phe

210 215 220

Ser Arg Glu Lys Gln His Leu Phe Asp Leu Ser Asp Lys Asp Ser Phe

225 230 235 240

Phe Asp Ser Lys Thr Arg Ser Thr Ile Val Tyr Glu Ile Leu Lys Arg

245 250 255

Thr Thr Cys Thr Lys Ala Lys Tyr Ser Met Gly Ile Thr Ser Leu Leu

260 265 270

Ala Asn Gly Val Tyr Ala Ala Ala Tyr Pro Leu His Asp Gly Asp Tyr

275 280 285

Asn Gly Glu Asn Val Glu Phe Asn Asp Arg Lys Leu Leu Tyr Glu Glu

290 295 300

Trp Ala Arg Tyr Gly Val Phe Tyr Lys Tyr Gln Pro Ile Asp Leu Val

305 310 315 320

Arg Lys Tyr Phe Gly Glu Lys Ile Gly Leu Tyr Phe Ala Trp Leu Gly

325 330 335

Val Tyr Thr Gln Met Leu Ile Pro Ala Ser Ile Val Gly Ile Ile Val

340 345 350

Phe Leu Tyr Gly Cys Ala Thr Met Asp Glu Asn Ile Pro Ser Met Glu

355 360 365

Met Cys Asp Gln Arg His Asn Ile Thr Met Cys Pro Leu Cys Asp Lys

370 375 380

Thr Cys Ser Tyr Trp Lys Met Ser Ser Ala Cys Ala Thr Ala Arg Ala

385 390 395 400

Ser His Leu Phe Asp Asn Pro Ala Thr Val Phe Phe Ser Val Phe Met

405 410 415

Ala Leu Trp Ala Ala Thr Phe Met Glu His Trp Lys Arg Lys Gln Met

420 425 430

Arg Leu Asn Tyr Arg Trp Asp Leu Thr Gly Phe Glu Glu Glu Glu Glu

435 440 445

Ala Val Lys Asp His Pro Arg Ala Glu Tyr Glu Ala Arg Val Leu Glu

450 455 460

Lys Ser Leu Lys Lys Glu Ser Arg Asn Lys Glu Lys Arg Arg His Ile

465 470 475 480

Pro Glu Glu Ser Thr Asn Lys Trp Lys Gln Arg Val Lys Thr Ala Met

485 490 495

Ala Gly Val Lys Leu Thr Asp Lys Val Lys Leu Thr Trp Arg Asp Arg

500 505 510

Phe Pro Ala Tyr Leu Thr Asn Leu Val Ser Ile Ile Phe Met Ile Ala

515 520 525

Val Thr Phe Ala Ile Val Leu Gly Val Ile Ile Tyr Arg Ile Ser Met

530 535 540

Ala Ala Ala Leu Ala Met Asn Ser Ser Pro Ser Val Arg Ser Asn Ile

545 550 555 560

Arg Val Thr Val Thr Ala Thr Ala Val Ile Ile Asn Leu Val Val Ile

565 570 575

Ile Leu Leu Asp Glu Val Tyr Gly Cys Ile Ala Arg Trp Leu Thr Lys

580 585 590

Ile Glu Val Pro Lys Thr Glu Lys Ser Phe Glu Glu Arg Leu Ile Phe

595 600 605

Lys Ala Phe Leu Leu Lys Phe Val Asn Ser Tyr Thr Pro Ile Phe Tyr

610 615 620

Val Ala Phe Phe Lys Gly Arg Phe Val Gly Arg Pro Gly Asp Tyr Val

625 630 635 640

Tyr Ile Phe Arg Ser Phe Arg Met Glu Glu Cys Ala Pro Gly Gly Cys

645 650 655

Leu Met Glu Leu Cys Ile Gln Leu Ser Ile Ile Met Leu Gly Lys Gln

660 665 670

Leu Ile Gln Asn Asn Leu Phe Glu Ile Gly Ile Pro Lys Met Lys Lys

675 680 685

Leu Ile Arg Tyr Leu Lys Leu Lys Gln Gln Ser Pro Pro Asp His Glu

690 695 700

Glu Cys Val Lys Arg Lys Gln Arg Tyr Glu Val Asp Tyr Asn Leu Glu

705 710 715 720

Pro Phe Ala Gly Leu Thr Pro Glu Tyr Met Glu Met Ile Ile Gln Phe

725 730 735

Gly Phe Val Thr Leu Phe Val Ala Ser Phe Pro Leu Ala Pro Leu Phe

740 745 750

Ala Leu Leu Asn Asn Ile Ile Glu Ile Arg Leu Asp Ala Lys Lys Phe

755 760 765

Val Thr Glu Leu Arg Arg Pro Val Ala Val Arg Ala Lys Asp Ile Gly

770 775 780

Ile Trp Tyr Asn Ile Leu Arg Gly Ile Gly Lys Leu Ala Val Ile Ile

785 790 795 800

Asn Ala Phe Val Ile Ser Phe Thr Ser Asp Phe Ile Pro Arg Leu Val

805 810 815

Tyr Leu Tyr Met Tyr Ser Lys Asn Gly Thr Met His Gly Phe Val Asn

820 825 830

His Thr Leu Ser Ser Phe Asn Val Ser Asp Phe Gln Asn Gly Thr Ala

835 840 845

Pro Asn Asp Pro Leu Asp Leu Gly Tyr Glu Val Gln Ile Cys Arg Tyr

850 855 860

Lys Asp Tyr Arg Glu Pro Pro Trp Ser Glu Asn Lys Tyr Asp Ile Ser

865 870 875 880

Lys Asp Phe Trp Ala Val Leu Ala Ala Arg Leu Ala Phe Val Ile Val

885 890 895

Phe Gln Asn Leu Val Met Phe Met Ser Asp Phe Val Asp Trp Val Ile

900 905 910

Pro Asp Ile Pro Lys Asp Ile Ser Gln Gln Ile His Lys Glu Lys Val

915 920 925

Leu Met Val Glu Leu Phe Met Arg Glu Glu Gln Asp Lys Gln Gln Leu

930 935 940

Leu Glu Thr Trp Met Glu Lys Glu Arg Gln Lys Asp Glu Pro Pro Cys

945 950 955 960

Asn His His Asn Thr Lys Ala Cys Pro Asp Ser Leu Gly Ser Pro Ala

965 970 975

Pro Ser His Ala Tyr His Gly Gly Val Leu

980 985

<210> 106

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 106

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Ser Gly

100 105 110

Thr Pro Val Thr Val Ser Ser Ala

115 120

<210> 107

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 107

Asp Ile Glu Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu

65 70 75 80

Asp Asp Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr

85 90 95

Phe Gly Ser Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 108

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 108

Gly Tyr Ser Phe Thr Gly Tyr

15

<210> 109

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 109

Thr Pro Tyr Asn Gly Ala

15

<210> 110

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 110

Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr

1 5 10

<210> 111

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 111

Ser Ser Val Ser Tyr Met His

15

<210> 112

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 112

Asp Thr Ser Lys Leu Ala Ser

15

<210> 113

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 113

Gln Gln Trp Ser Lys His Pro Leu Thr

15

<210> 114

<211> 131

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 114

Glu Val His Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Gln Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Lys Gln Ala Gly Ser Glu Lys Thr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Ala Tyr Tyr Tyr Asp Ser Ala Ser Tyr Tyr Pro Tyr

100 105 110

Tyr Tyr Tyr Tyr Ser Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr

115 120 125

Val Ser Ser

130

<210> 115

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 115

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Gln

85 90 95

Tyr Thr Phe Gly Glyn Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 116

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 116

Gly Phe Thr Phe Ser Arg Tyr

15

<210> 117

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 117

Lys Gln Ala Gly Ser Glu

15

<210> 118

<211> 22

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 118

Glu Gly Ala Tyr Tyr Tyr Asp Ser Ala Ser Tyr Tyr Pro Tyr Tyr Tyr

1 5 10 15

Tyr Tyr Ser Met Asp Val

20

<210> 119

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 119

Gln Ser Val Ser Ser Ser Tyr Leu Ala

15

<210> 120

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 120

Gly Ala Ser Ser Arg Ala Thr

15

<210> 121

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 121

Gln Gln Tyr Gly Ser Ser Gln Tyr Thr

15

<210> 122

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 122

Gln Val Glu Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asp Pro Gly Asp Ser Arg Thr Arg Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gln Leu Tyr Gly Gly Thr Tyr Met Asp Gly Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala

115 120

<210> 123

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 123

Asp Ile Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr

20 25 30

Asn Ser Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Gly Val Asn Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Asp Ile Glu

85 90 95

Ser Ala Thr Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

<210> 124

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 124

Gly Tyr Ser Phe Thr Ser Tyr

15

<210> 125

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 125

Asp Pro Gly Asp Ser Arg

15

<210> 126

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 126

Gly Glyn Leu Tyr Gly Gly Thr Tyr Met Asp Gly

1 5 10

<210> 127

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 127

Ser Ser Asp Ile Gly Gly Tyr Asn Ser Val Ser

1 5 10

<210> 128

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 128

Gly Val Asn Asn Arg Pro Ser

15

<210> 129

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 129

Ser Ser Tyr Asp Ile Glu Ser Ala Thr Pro Val

1 5 10

<210> 130

<211> 630

<212> Protein

<213> Unknown

<220>

<223> Description of unknown:

mesothelin polypeptide

<400> 130

Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro

1 5 10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln

20 25 30

Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu

35 40 45

Asp Gly Val Leu Ala Asn Pro Asn Ile Ser Ser Leu Ser Pro Arg

50 55 60

Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu

65 70 75 80

Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu

85 90 95

Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro

100 105 110

Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro

115 120 125

Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile

130 135 140

Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln

145 150 155 160

Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu

165 170 175

Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu

180 185 190

Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu

195 200 205

Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg

210 215 220

Ala Ala Leu Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp

225 230 235 240

Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly

245 250 255

Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg

260 265 270

Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile

275 280 285

Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser

290 295 300

Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys

305 310 315 320

Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met

325 330 335

Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu

340 345 350

Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val

355 360 365

Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile

370 375 380

Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu

385 390 395 400

Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu

405 410 415

Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln

420 425 430

Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr

435 440 445

Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser

450 455 460

Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln

465 470 475 480

Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn

485 490 495

Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro

500 505 510

Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu

515 520 525

Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val

530 535 540

Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala

545 550 555 560

Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln

565 570 575

Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn

580 585 590

Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr

595 600 605

Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu

610 615 620

Leu Ala Ser Thr Leu Ala

625 630

<210> 131

<211> 246

<212> Protein

<213> Unknown

<220>

<223> Description of unknown:

ULBP-6 polypeptide

<400> 131

Met Ala Ala Ala Ala Ile Pro Ala Leu Leu Leu Cys Leu Pro Leu Leu

1 5 10 15

Phe Leu Leu Phe Gly Trp Ser Arg Ala Arg Arg Asp Asp Pro His Ser

20 25 30

Leu Cys Tyr Asp Ile Thr Val Ile Pro Lys Phe Arg Pro Gly Pro Arg

35 40 45

Trp Cys Ala Val Gln Gly Gln Val Asp Glu Lys Thr Phe Leu His Tyr

50 55 60

Asp Cys Gly Asn Lys Thr Val Thr Pro Val Ser Pro Leu Gly Lys Lys

65 70 75 80

Leu Asn Val Thr Met Ala Trp Lys Ala Gln Asn Pro Val Leu Arg Glu

85 90 95

Val Val Asp Ile Leu Thr Glu Gln Leu Leu Asp Ile Gln Leu Glu Asn

100 105 110

Tyr Thr Pro Lys Glu Pro Leu Thr Leu Gln Ala Arg Met Ser Cys Glu

115 120 125

Gln Lys Ala Glu Gly His Ser Ser Gly Ser Trp Gln Phe Ser Ile Asp

130 135 140

Gly Gln Thr Phe Leu Leu Phe Asp Ser Glu Lys Arg Met Trp Thr Thr

145 150 155 160

Val His Pro Gly Ala Arg Lys Met Lys Glu Lys Trp Glu Asn Asp Lys

165 170 175

Asp Val Ala Met Ser Phe His Tyr Ile Ser Met Gly Asp Cys Ile Gly

180 185 190

Trp Leu Glu Asp Phe Leu Met Gly Met Asp Ser Thr Leu Glu Pro Ser

195 200 205

Ala Gly Ala Pro Leu Ala Met Ser Ser Gly Thr Thr Gln Leu Arg Ala

210 215 220

Thr Ala Thr Thr Leu Ile Leu Cys Cys Leu Leu Ile Ile Leu Pro Cys

225 230 235 240

Phe Ile Leu Pro Gly Ile

245

<210> 132

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 132

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 133

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 133

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asp Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 134

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 134

Phe Thr Phe Ser Ser Tyr Ala Met Ser

15

<210> 135

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 135

Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 136

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 136

Ala Lys Asp Gly Gly Tyr Tyr Asp Ser Gly Ala Gly Asp Tyr

1 5 10

<210> 137

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 137

Arg Ala Ser Gln Gly Ile Asp Ser Trp Leu Ala

1 5 10

<210> 138

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 138

Ala Ala Ser Ser Leu Gln Ser

15

<210> 139

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 139

Gln Gln Gly Val Ser Tyr Pro Arg Thr

15

<210> 140

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 140

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 141

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 141

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 142

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 142

Phe Thr Phe Ser Ser Tyr Ser Met Asn

15

<210> 143

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 143

Ser Ile Ser Ser Ser Ser Ser Ser Tyr Ile Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 144

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 144

Ala Arg Gly Ala Pro Met Gly Ala Ala Ala Gly Trp Phe Asp Pro

1 5 10 15

<210> 145

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 145

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 146

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 146

Ala Ala Ser Ser Leu Gln Ser

15

<210> 147

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 147

Gln Gln Gly Val Ser Phe Pro Arg Thr

15

<210> 148

<211> 124

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 148

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 149

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 149

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Asp Asp Tyr Trp Pro Pro

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 150

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 150

Tyr Thr Phe Thr Gly Tyr Tyr Met His

15

<210> 151

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 151

Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 152

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 152

Ala Arg Asp Thr Gly Glu Tyr Tyr Asp Thr Asp Asp His Gly Met Asp

1 5 10 15

Val

<210> 153

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 153

Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala

1 5 10

<210> 154

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 154

Gly Ala Ser Thr Arg Ala Thr

15

<210> 155

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 155

Gln Gln Asp Asp Tyr Trp Pro Pro Thr

15

<210> 156

<211> 126

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 156

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Pro Asn Tyr Gly Asp Thr Thr His Asp Tyr Tyr Tyr

100 105 110

Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 157

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 157

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Asp Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 158

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 158

Tyr Thr Phe Thr Ser Tyr Tyr Met His

15

<210> 159

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 159

Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln

1 5 10 15

gly

<210> 160

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 160

Ala Arg Gly Ala Pro Asn Tyr Gly Asp Thr Thr His Asp Tyr Tyr Tyr

1 5 10 15

Met Asp Val

<210> 161

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 161

Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala

1 5 10

<210> 162

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 162

Gly Ala Ser Thr Arg Ala Thr

15

<210> 163

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 163

Gln Gln Tyr Asp Asp Trp Pro Phe Thr

15

<210> 164

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 164

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 165

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 165

Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Thr Phe Ile Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 166

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 166

Gly Ser Phe Ser Gly Tyr Tyr Trp Ser

15

<210> 167

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 167

Glu Ile Asp His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 168

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 168

Ala Arg Ala Arg Gly Pro Trp Ser Phe Asp Pro

1 5 10

<210> 169

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 169

Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala

1 5 10

<210> 170

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 170

Lys Ala Ser Ser Leu Glu Ser

15

<210> 171

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 171

Gln Gln Tyr Asp Thr Phe Ile Thr

15

<210> 172

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 172

Ser Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Thr Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Ser Leu Val Thr Val Ser Ser

115 120

<210> 173

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 173

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 174

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 174

Gly Tyr Thr Phe Thr Asn Tyr

15

<210> 175

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 175

Asn Thr Tyr Thr Gly Glu

15

<210> 176

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 176

Gly Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val

1 5 10

<210> 177

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 177

Gln Asp Val Ser Ile Ala Val Ala

15

<210> 178

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 178

Ser Ala Ser Tyr Arg Tyr Thr

15

<210> 179

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 179

Gln Gln His Tyr Ile Thr Pro Leu Thr

15

<210> 180

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 180

Gln Ile Gln Leu Val Gln Ser Gly His Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Met Tyr Phe Cys

85 90 95

Gly Arg Gly Gly Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 181

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 181

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 182

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 182

Gly Tyr Thr Phe Thr Asn Tyr

15

<210> 183

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 183

Asn Thr Lys Thr Gly Glu

15

<210> 184

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 184

Gly Gly Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val

1 5 10

<210> 185

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 185

Gln Asp Val Ser Ile Ala Val Ala

15

<210> 186

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 186

Ser Ala Ser Tyr Arg Tyr Thr

15

<210> 187

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 187

Gln Gln His Tyr Ile Thr Pro Leu Thr

15

<210> 188

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 188

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala

20 25 30

Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe

50 55 60

Lys Gly Arg Val Thr Ile Ser Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 189

<211> 109

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 189

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr

100 105

<210> 190

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 190

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala

20 25 30

Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe

50 55 60

Lys Gly Arg Val Thr Ile Ser Leu Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 191

<211> 109

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 191

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr

100 105

<210> 192

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 192

Gln Ile Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala

20 25 30

Gly Met Gln Trp Val Gln Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe

50 55 60

Lys Gly Arg Val Thr Phe Ser Leu Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Phe Gly Ser Ser Tyr Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 193

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 193

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Gln Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln His Tyr Ile Thr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 194

<211> 323

<212> Protein

<213> Unknown

<220>

<223> Description of unknown:

TROP2 polypeptide

<400> 194

Met Ala Arg Gly Pro Gly Leu Ala Pro Pro Pro Leu Arg Leu Pro Leu

1 5 10 15

Leu Leu Leu Val Leu Ala Ala Val Thr Gly His Thr Ala Ala Gln Asp

20 25 30

Asn Cys Thr Cys Pro Thr Asn Lys Met Thr Val Cys Ser Pro Asp Gly

35 40 45

Pro Gly Gly Arg Cys Gln Cys Arg Ala Leu Gly Ser Gly Met Ala Val

50 55 60

Asp Cys Ser Thr Leu Thr Ser Lys Cys Leu Leu Leu Lys Ala Arg Met

65 70 75 80

Ser Ala Pro Lys Asn Ala Arg Thr Leu Val Arg Pro Ser Glu His Ala

85 90 95

Leu Val Asp Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Pro Glu Gly

100 105 110

Arg Phe Lys Ala Arg Gln Cys Asn Gln Thr Ser Val Cys Trp Cys Val

115 120 125

Asn Ser Val Gly Val Arg Arg Thr Asp Lys Gly Asp Leu Ser Leu Arg

130 135 140

Cys Asp Glu Leu Val Arg Thr His His Ile Leu Ile Asp Leu Arg His

145 150 155 160

Arg Pro Thr Ala Gly Ala Phe Asn His Ser Asp Leu Asp Ala Glu Leu

165 170 175

Arg Arg Leu Phe Arg Glu Arg Tyr Arg Leu His Pro Lys Phe Val Ala

180 185 190

Ala Val His Tyr Glu Gln Pro Thr Ile Gln Ile Glu Leu Arg Gln Asn

195 200 205

Thr Ser Gln Lys Ala Ala Gly Asp Val Asp Ile Gly Asp Ala Ala Tyr

210 215 220

Tyr Phe Glu Arg Asp Ile Lys Gly Glu Ser Leu Phe Gln Gly Arg Gly

225 230 235 240

Gly Leu Asp Leu Arg Val Arg Gly Glu Pro Leu Gln Val Glu Arg Thr

245 250 255

Leu Ile Tyr Tyr Leu Asp Glu Ile Pro Pro Lys Phe Ser Met Lys Arg

260 265 270

Leu Thr Ala Gly Leu Ile Ala Val Ile Val Val Val Val Val Ala Leu

275 280 285

Val Ala Gly Met Ala Val Leu Val Ile Thr Asn Arg Arg Lys Ser Gly

290 295 300

Lys Tyr Lys Lys Val Glu Ile Lys Glu Leu Gly Glu Leu Arg Lys Glu

305 310 315 320

Pro Ser Leu

<210> 195

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 195

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Asp Phe Thr Thr Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile His Pro Asp Ser Ser Thr Ile Asn Tyr Ala Pro Ser Leu

50 55 60

Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys

85 90 95

Ala Ser Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Pro Val Thr Val Ser Ser

115

<210> 196

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 196

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ser

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Leu Tyr Arg Ser

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 197

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 197

Gly Phe Asp Phe Thr Thr Tyr

15

<210> 198

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 198

His Pro Asp Ser Ser Thr

15

<210> 199

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 199

Leu Tyr Phe Gly Phe Pro Trp Phe Ala Tyr

1 5 10

<210> 200

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 200

Gln Asp Val Gly Thr Ser Val Ala

15

<210> 201

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 201

Trp Thr Ser Thr Arg His Thr

15

<210> 202

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 202

Gln Gln Tyr Ser Leu Tyr Arg Ser

15

<210> 203

<211> 122

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 203

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala

115 120

<210> 204

<211> 109

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 204

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 205

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 205

Gly Tyr Thr Phe Thr Glu Phe

15

<210> 206

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 206

Asn Thr Lys Thr Gly Glu

15

<210> 207

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 207

Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr

1 5 10

<210> 208

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 208

Ala Ala Val Gly Thr Tyr Val Ala

15

<210> 209

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 209

Ser Ala Ser Tyr Arg Lys Arg

15

<210> 210

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 210

His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr

1 5 10

<210> 211

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 211

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala

1 5 10 15

Ser Ile Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys His Tyr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asn Pro Glu Asn Val Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn His Tyr Arg Tyr Ala Gly Gly Gly Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala

115 120

<210> 212

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 212

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Val Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Thr Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Asn Asn Pro Tyr Ser

85 90 95

Phe Gly Gly Gly Thr Lys Val Val Ile Lys Thr Val

100 105

<210> 213

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 213

Gly Phe Asn Ile Lys His Tyr

15

<210> 214

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 214

Asn Pro Glu Asn Val Asp

15

<210> 215

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 215

Tyr Arg Tyr Ala Gly Gly Gly Ala Leu Asp Tyr

1 5 10

<210> 216

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 216

Ser Ser Val Ser Tyr Ile His

15

<210> 217

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 217

Asp Thr Ser Lys Leu Ala Ser

15

<210> 218

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 218

Gln Gln Trp Asn Asn Asn Pro Tyr Ser

15

<210> 219

<211> 702

<212> Protein

<213> Unknown

<220>

<223> Description of unknown:

CEA polypeptide

<400> 219

Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln

1 5 10 15

Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile

100 105 110

Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

195 200 205

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

210 215 220

Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser

290 295 300

Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala

305 310 315 320

Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu

325 330 335

Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr

340 345 350

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg

355 360 365

Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr

370 375 380

Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser

385 390 395 400

Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp

405 410 415

Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn

420 425 430

Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser

435 440 445

Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile

450 455 460

Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn

465 470 475 480

Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val

485 490 495

Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro

500 505 510

Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln

515 520 525

Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser

530 535 540

Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn

545 550 555 560

Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser

565 570 575

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

580 585 590

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

595 600 605

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

610 615 620

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

625 630 635 640

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

645 650 655

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

660 665 670

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

675 680 685

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile

690 695 700

<210> 220

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 220

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 221

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 221

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 222

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 222

Gly Tyr Thr Phe Thr Asn Tyr Gly

15

<210> 223

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 223

Ile Asn Thr Asn Thr Gly Glu Pro

15

<210> 224

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 224

Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr

1 5 10

<210> 225

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 225

Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

1 5 10

<210> 226

<211> 3

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 226

Trp Ala Ser

1

<210> 227

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 227

Gln Asn Asp Tyr Ser Tyr Pro Leu Thr

15

<210> 228

<211> 118

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 228

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 229

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 229

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 230

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 230

Gly Tyr Thr Phe Thr Ser Tyr Trp

15

<210> 231

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 231

Ile Tyr Pro Ser Asp Ser Tyr Thr

15

<210> 232

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 232

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr

1 5 10

<210> 233

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 233

Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr

1 5 10

<210> 234

<211> 3

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 234

Trp Ala Ser

1

<210> 235

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 235

Gln Asn Asp Tyr Ser Tyr Pro Phe Thr

15

<210> 236

<211> 261

<212> Protein

<213> Unknown

<220>

<223> Description of unknown:

Claudin-18.2 polypeptide

<400> 236

Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile

1 5 10 15

Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

115 120 125

Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

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Claims (30)

1. A polyspecific binding protein that binds NKG2D, CEA and CD16, containing: (a) a first antigen-binding site comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) of an anti-NKG2D antibody; (b) a second antigen-binding site containing VH and VL antibodies to CEA; And (c) the first Fc-domain polypeptide of the antibody, or a portion thereof; And (d) a second antibody Fc-domain polypeptide or a portion thereof, wherein said first antibody Fc domain polypeptide or portion thereof and said second antibody Fc domain polypeptide or portion thereof together bind CD16, and wherein said VH or VL of an anti-NKG2D antibody is fused to the N-terminus of said first antibody Fc-domain polypeptide or portion thereof, and said VH or VL of an anti-CEA antibody is fused to the N-terminus of said second antibody Fc-domain polypeptide or portion thereof. 2. The polyspecific binding protein that binds NKG2D, CEA and CD16 according to claim 1, wherein the VH and VL of the first antigen binding site bind to NKG2D in humans, non-human primates and rodents. 3. A polyspecific binding protein that binds NKG2D, CEA and CD16 according to claim 1 or 2, wherein the VH and VL of the first antigen binding site are present in the same polypeptide. 4. The polyspecific binding protein that binds NKG2D, CEA and CD16 according to claim 1, wherein the VH and VL of the second antigen binding site are present in the same polypeptide. 5. The polyspecific binding protein that binds NKG2D, CEA and CD16 according to claim 4, wherein the VL of the first antigen binding site has an amino acid sequence identical to the amino acid sequence of the VL of the second antigen binding site. 6. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1 to 5, wherein the VH of the first antigen binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 1. 7. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-5, wherein the VH of the first antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 41, and the VL of the first antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 42. 8. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-5, wherein the VH of the first antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 43, and the VL of the first antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 44. 9. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-5, wherein the VH of the first antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 45, and the VL of the first antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 46. 10. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-5, wherein the VH of the first antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 47, and the VL of the first antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 48. 11. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-5, where the VH of the first antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 49, and the VL of the first antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 50. 12. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1 to 5, wherein the VH of the first antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 132, and the VL of the first antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 133. 13. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-12, where the VH of the second antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 195, and the VL of the second antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 196. 14. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-12, where the VH of the second antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 203, and the VL of the second antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 204. 15. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1-12, where the VH of the second antigen-binding site contains an amino acid sequence at least 90% identical to SEQ ID NO: 211, and the VL of the second antigen-binding site The site contains an amino acid sequence at least 90% identical to SEQ ID NO: 212. 16. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 1 to 15, wherein the Fc domain of the antibody contains the hinge and CH2 domains. 17. A polyspecific binding protein that binds NKG2D, CEA, and CD16, according to claim 16, wherein the Fc domain of the antibody contains the hinge and CH2 domains of a human IgG1 antibody. 18. A polyspecific binding protein that binds NKG2D, CEA, and CD16, according to claim 16 or 17, wherein the Fc domain of the antibody contains an amino acid sequence at least 90% identical to amino acids 234-332 of a human IgG1 antibody. 19. A polyspecific binding protein that binds NKG2D, CEA and CD16, according to any one of claims 16-18, where the Fc domain of the antibody contains an amino acid sequence at least 90% identical to the Fc domain of human IgG1 and differs in one or more positions selected from the group consisting of Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409 , T411 and K439. 20. A pharmaceutical composition for the treatment of cancer, comprising an effective amount of a polyspecific binding protein according to any one of claims 1-19 and a pharmaceutically acceptable carrier. 21. A host cell for obtaining a polyspecific binding protein that binds NKG2D, CEA and CD16, containing one or more nucleic acids expressing a polyspecific binding protein according to any one of claims 1-19. 22. A method for directly or indirectly enhancing the death of tumor cells, including the effect of a polyspecific binding protein according to any one of claims 1-19 on a tumor and on natural killer cells. 23. A method of treating a malignant neoplasm, wherein said method comprises administering to a patient a polyspecific binding protein according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20. 24. The method of claim 23, wherein the cancer to be treated is selected from the group consisting of gastrointestinal cancer, colorectal cancer, pancreatic cancer, non-small cell lung cancer, and breast cancer.

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