Multi-chain chimeric polypeptide and use of the same

Abstract

To provide a multi-chain chimeric polypeptide and use thereof.SOLUTION: Provided is a multi-chain chimeric polypeptide that includes (a) a first chimeric polypeptide including a first target binding domain, a soluble tissue factor domain and a first domain of a pair of affinity domains, and (b) a second chimeric polypeptide including a second domain of a pair of affinity domains and a second target binding domain, where the first chimeric polypeptide and the second chimeric polypeptide are aggregated via a bond between the first domain and the second domain of the pair of affinity domains. Also provided is a method of using the multi-chain chimeric polypeptides, and a nucleic acid encoding the multi-chain chimeric polypeptides.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is incorporated herein by reference in its entirety, including U.S. Patent Application Nos. 62 / 724,969, filed August 30, 2018; 62 / 817,230, filed March 12, 2019; 62 / 725,043, filed August 30, 2018; 62 / 725,010, filed August 30, 2018; and 62 / 749, filed October 22, 2018. ,007, filed October 17, 2018, U.S. Patent Application No. 62 / 746,832, filed October 23, 2018, U.S. Patent Application No. 62 / 749,506, filed October 23, 2018, U.S. Patent Application No. 62 / 817,241, filed March 12, 2019, U.S. Patent Application No. 62 / 816,683, filed March 11, 2019, and U.S. Patent Application No. 62 / 881,088, filed July 31, 2019.

[0002] Technical Field The present disclosure relates to the field of biotechnology, and more specifically to antigen-binding molecules. [Background technology]

[0003] Tissue factor (TF), a 263-amino acid integral membrane glycoprotein with a molecular weight of approximately 46 kDa and the trigger protein of the extrinsic blood coagulation pathway, is the primary initiator of coagulation in vivo. Normally, tissue factor, not in contact with circulating blood, initiates the coagulation cascade upon exposure to circulating serine protease factors. Vascular injury exposes subendothelial cells expressing tissue factor, resulting in the formation of a calcium-dependent, high-affinity complex with pre-existing plasma factor VIIa (FVIIa). Binding of the serine protease FVIIa to tissue factor promotes rapid cleavage of FX to FXa and FIX to FIXa. The resulting proteolytic activity of FXa and the active membrane surface then inefficiently converts small amounts of prothrombin to thrombin. Thrombin generated by FXa initiates platelet activation, activating trace amounts of procofactors factor V (FV) and factor VIII (FVIII) to the active cofactors factor Va (FVa) and factor VIIIa (FVIIIa). FIXa complexes with FVIIIa on the platelet surface to form the intrinsic tenase complex, which leads to the rapid generation of FXa, and FXa complexes with FVa to form the prothrombinase complex on the surface of activated platelets, which leads to the rapid cleavage of prothrombin to thrombin.

[0004] In addition to the tissue factor-FVIIa complex, recent studies have shown that the tissue factor-FVIIa-FXa complex can activate FVIII, thereby providing additional levels of FVIIIa during the initiation phase. While the extrinsic pathway is most important in initiating coagulation by activating a limited amount of thrombin, the intrinsic pathway sustains coagulation by dramatically amplifying the initial signal.

[0005] Much of the tissue factor expressed on the cell surface is "encoded" and must be "decoded" to fully participate in coagulation. The mechanism by which cell surface tissue factor is "decoded" remains unclear at present, but exposure of anionic phospholipids plays a major role in this process. Healthy cells actively sequester anionic phospholipids, such as phosphatidylserine (PS), in the inner leaflet of the plasma membrane. Upon cell injury, activation, or cytoplasmic Ca 2+ After increased levels, this bilayer asymmetry is lost, resulting in increased PS exposure in the outer leaflet, which increases the specific activity of cell surface tissue factor-FVIIa complexes. PS exposure reduces the apparent Km for activation of FIX and FX by tissue factor-FVIIa complexes. Although known, additional mechanisms may involve conformational rearrangements of tissue factor or tissue factor-FVIIa and subsequent exposure of the substrate-binding site. Summary of the Invention

[0006] The present invention is based on the discovery that soluble tissue factor can be used as a scaffold for chimeric polypeptides comprising antigen-binding domains. Based on this discovery, provided herein is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising (i) a first target-binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising (i) a second domain of the pair of affinity domains and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated via the binding between the first domain and the second domain of the pair of affinity domains. Also provided herein are compositions comprising any of the multi-chain chimeric polypeptides described herein, nucleic acids encoding any of the multi-chain chimeric polypeptides described herein, and cells comprising any of the nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods for stimulating immune cells and methods for treating a subject in need of treatment, comprising the use of any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods for producing any of the multi-chain chimeric polypeptides described herein.

[0007] therefore, (a)(i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains; a first chimeric polypeptide comprising: (b)(i) a second domain of the pair of affinity domains; (ii) a second target-binding domain; and a second chimeric polypeptide comprising Provided herein are multi-chain chimeric polypeptides comprising: a first chimeric polypeptide and a second chimeric polypeptide associated via binding between a first domain and a second domain of a pair of affinity domains; in some embodiments, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide; in some embodiments, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide; in some embodiments, the soluble tissue factor domain and the first domain of the pair of affinity domains are immediately adjacent to each other within the first chimeric polypeptide; in some embodiments, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains within the first chimeric polypeptide; in some embodiments, the second domain of the pair of affinity domains and the second target binding domain are immediately adjacent to each other within the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between the second domain and the second target binding domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments, the first target binding domain and the second target binding domain comprise the same amino acid sequence. In some embodiments, the first target binding domain and the second target binding domain specifically bind to different antigens. In some embodiments, one or both of the first target binding domain and the second target binding domain are antigen binding domains. In some embodiments, the first target binding domain and the second target binding domain are each antigen binding domains. In some embodiments, the antigen binding domain comprises an scFv or a single domain antibody. In some embodiments, one or both of the first target binding domain and the second target binding domain are selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, The first and second target binding domains specifically bind to a target selected from the group consisting of TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor. In some embodiments, one or both of the first and second target binding domains is a soluble interleukin, a soluble cytokine protein, or a ligand protein. In some embodiments, the soluble interleukin, soluble cytokine, or ligand protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.In some embodiments, one or both of the first target binding domain and the second target binding domain are soluble interleukin, cytokine receptor, or soluble cell surface receptor. In some embodiments, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28. In some embodiments, the first chimeric polypeptide further comprises one or more additional target binding domain(s), and at least one of the one or more additional antigen binding domain(s) is / are located between the soluble tissue factor domain and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and at least one of the one or more additional antigen binding domain(s) and / or between at least one of the one or more additional antigen binding domain(s) and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains is immediately adjacent to the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains. In some embodiments, at least one of the one or more additional target binding domains is immediately adjacent to the first target binding domain in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains.In some embodiments, at least one of the one or more additional target binding domains is located at the N-terminus and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains is paired with a soluble tissue factor domain within the first chimeric polypeptide. In some embodiments, at least one additional target binding domain of the one or more additional target binding domains disposed at the N-terminus is immediately adjacent to the target binding domain in the first chimeric polypeptide or the first domain of the first pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains. In some embodiments, at least one additional target binding domain of the one or more additional target binding domains disposed at the C-terminus is immediately adjacent to the target binding domain in the first chimeric polypeptide or the first domain of the first pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of the pair of affinity domains. In some embodiments, at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains is immediately adjacent to the soluble tissue factor domain and / or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed between (i) the soluble tissue factor domain and at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains, and / or (ii) between the first domain of the pair of affinity domains and at least one of the one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains. In some embodiments, the second chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.In some embodiments, at least one of the one or more additional target binding domains is immediately adjacent to the second domain of the pair of affinity domains in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain of the pair of affinity domains. In some embodiments, at least one of the one or more additional target binding domains is immediately adjacent to the second target binding domain in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to different antigens. In some embodiments, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains is an antigen binding domain.In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each an antigen binding domain. In some embodiments, the antigen binding domain comprises an scFv. In some embodiments, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand , scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, CD3 receptor, and CD28 receptor. In some embodiments, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains are soluble interleukins, soluble cytokine proteins, or ligand proteins. In some embodiments, the soluble interleukin, soluble cytokine, or ligand protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. In some embodiments, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains are soluble interleukin or cytokine receptors.In some embodiments, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28. In some embodiments, the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO:1. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO:1. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not comprise one or more of: a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein, an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein, a tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, an arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and a phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein ... The soluble tissue factor domain does not contain any of the following: lysine; isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein; tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein; aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein; tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein; arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein; and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain is unable to bind to factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments, the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal. In some embodiments of any of the single-chain chimeric polypeptides provided herein, the human soluble tissue factor domain does not initiate blood clotting. In some embodiments of any of the single-chain chimeric polypeptides provided herein, the soluble tissue factor domain comprises or consists of soluble wild-type human tissue factor.

[0008] In some embodiments, the pair of affinity domains is a sushi domain from human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. In some embodiments, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments, the human IL15Rα is mature, full-length IL15Rα. In some embodiments, the pair of affinity domains is selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter / docking tag modules based on mutant RNase I fragments, and SNARE modules based on the interactions of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide further comprise a signal sequence at its N-terminus.

[0009] In some embodiments, the multi-chain chimeric polypeptide comprises a composition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises at least one dose of the multi-chain chimeric polypeptide. In some embodiments, a kit comprises at least one dose of the composition.

[0010] In some embodiments, provided herein are methods for stimulating immune cells, the method comprising contacting the immune cells with an effective amount of any of the multi-chain chimeric polypeptides or compositions described above. In some embodiments, the method comprises contacting the immune cells in vitro. In some embodiments, the method comprises obtaining the immune cells from a subject. In some embodiments, the method comprises obtaining the immune cells from a subject prior to the contacting step. In some embodiments, the method comprises contacting the immune cells in vivo. In some embodiments, the method comprises contacting the immune cells with immature thymocytes, peripheral blood lymphocytes, naive T cells, multipotent Th cell precursors, lymphoid progenitor cells, Treg cells, memory T cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8 + T cells, CD4 + The method comprises selecting the immune cells from the group consisting of T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells. In some embodiments, the method comprises genetically modifying the immune cells to express a chimeric antigen receptor or a recombinant T cell receptor. In some embodiments, the method comprises, after the contacting step, introducing into the immune cells a nucleic acid encoding the chimeric antigen receptor or the recombinant T cell receptor. In some embodiments, the method comprises administering the immune cells to a subject in need thereof. In some embodiments, the method comprises identifying or diagnosing the subject as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related lung tissue elasticity loss, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the method includes identifying or diagnosing the subject as having cancer. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method comprises diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

[0011] Also provided herein are methods for inducing or increasing the proliferation of immune cells, comprising contacting the immune cells with an effective amount of any of the multi-chain chimeric polypeptides or compositions described above. In some embodiments, the method comprises contacting the immune cells in vitro. In some embodiments, the method comprises obtaining the immune cells from a subject. In some embodiments, the method comprises obtaining the immune cells from a subject prior to the contacting step. In some embodiments, the method comprises contacting the immune cells in vivo. In some embodiments, the method comprises contacting immature thymocytes, peripheral blood lymphocytes, naive T cells, multipotent Th cell precursors, lymphoid progenitor cells, Treg cells, memory T cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8 + T cells, CD4 +The method comprises selecting the immune cells from the group consisting of T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells. In some embodiments, the method comprises modifying the immune cells to express a chimeric antigen receptor or a recombinant T cell receptor. In some embodiments, the method further comprises, after the contacting step, introducing a nucleic acid encoding the chimeric antigen receptor or recombinant T cell receptor into the immune cells. In some embodiments, the method further comprises administering the immune cells to a subject in need thereof. In some embodiments, the method comprises identifying or diagnosing the subject as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related lung tissue elasticity loss, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the method includes identifying or diagnosing the subject as having cancer. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method includes diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

[0012] Also provided herein are methods for inducing differentiation of immune cells into memory or memory-like immune cells, comprising contacting the immune cells with an effective amount of any of the multi-chain chimeric polypeptides or compositions described above. In some embodiments, the method comprises contacting the immune cells in vitro. In some embodiments, the method comprises obtaining the immune cells from a subject. In some embodiments, the method further comprises obtaining the immune cells from a subject prior to the contacting step. In some embodiments, the method comprises contacting the immune cells in vivo. In some embodiments, the immune cells are selected from the group consisting of immature thymocytes, peripheral blood lymphocytes, naive T cells, multipotent Th cell precursors, lymphoid progenitor cells, Treg cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8 + T cells, CD4 +The immune cells are selected from the group consisting of T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells. In some embodiments, the immune cells have been genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor. In some embodiments, the method further comprises, after the contacting step, introducing a nucleic acid encoding the chimeric antigen receptor or the recombinant T cell receptor into the immune cells. In some embodiments, the method further comprises administering the immune cells to a subject in need thereof. In some embodiments, the method comprises identifying or diagnosing the subject as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related lung tissue elasticity loss, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis and renal dysfunction.In some embodiments, this method comprises identifying or diagnosing the subject as having cancer. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.In some embodiments, the method includes diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is a human immune disease. These include infections with HIV, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

[0013] Also provided herein are methods for killing cancerous, infected, or senescent cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides or compositions described above. In some embodiments, the method comprises identifying or diagnosing the subject as having cancer. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method includes identifying or diagnosing the subject as having an aging-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related lung tissue elasticity loss, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.In some embodiments, the method comprises administering to a subject a therapeutically effective amount of any of the above-mentioned multi-chain chimeric polypeptides or compositions. In some embodiments, the method includes identifying or diagnosing the subject as having cancer.In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method includes identifying or diagnosing the subject as having an aging-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related lung tissue elasticity loss, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis and renal dysfunction.In some embodiments, this method comprises diagnosing or identifying the subject as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

[0014] Also provided herein are nucleic acids encoding any of the above-described multi-chain chimeric polypeptides. Some embodiments include vectors comprising any of the above-described nucleic acids. In some embodiments, the vector is an expression vector. Some embodiments include cells comprising any of the above-described nucleic acids.

[0015] Also provided herein are methods for producing a multi-chain chimeric polypeptide, comprising culturing cells in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide, and recovering the multi-chain chimeric polypeptide from the cells and / or the culture medium. In some embodiments, the methods comprise producing the multi-chain chimeric polypeptide by the methods described above.

[0016] In some embodiments, the variant soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO:3. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO:3. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO:3. In some embodiments, the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO:3. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO:4. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO:4. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO:4. In some embodiments, the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO:4.

[0017] As used herein, the term "chimeric" refers to a polypeptide that includes amino acid sequences (e.g., domains) that are originally derived from two different sources (e.g., two different naturally occurring proteins, from the same or different species). For example, a chimeric polypeptide can include domains that are derived from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can include a domain that is a synthetic sequence (e.g., an scFv) and a domain that is derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide can include at least two different domains that are synthetic sequences (e.g., two different scFvs).

[0018] An "antigen-binding domain" is one or more protein domain(s) (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigen(s). In some examples, an antigen-binding domain can bind to an antigen or epitope with similar specificity and affinity as a naturally occurring antibody. In some embodiments, the antigen-binding domain may be an antibody or fragment thereof. In some embodiments, the antigen-binding domain may comprise an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art.

[0019] A "soluble tissue factor domain" refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) with a segment of a wild-type mammalian tissue factor protein (e.g., a wild-type human tissue factor protein) lacking the transmembrane and intracellular domains. Non-limiting examples of soluble tissue factor domains are described herein.

[0020] The term "soluble interleukin protein" is used herein to refer to a mature secreted interleukin protein or a biologically active fragment thereof. In some examples, a soluble interleukin protein can comprise a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein) and retains its biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

[0021] The term "soluble cytokine protein" is used herein to refer to a mature secreted cytokine protein or a biologically active fragment thereof. In some examples, a soluble cytokine protein can comprise a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein) and retains its biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

[0022] The term "soluble interleukin receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that can bind to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble interleukin receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type interleukin receptor and retains the ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks the intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0023] The term "soluble cytokine receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble cytokine receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type cytokine receptor and retains the ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks the intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0024] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0025] "Affinity" refers to the strength of the sum of non-covalent interactions between an antigen-binding site and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, When used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between a member of an antigen-binding domain and an antigen or epitope. The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D ) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity of an antigen-binding domain for its corresponding antigen or epitope are known in the art.

[0026] As used herein, a "multi-chain polypeptide" refers to a polypeptide comprising two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), wherein the two or more protein chains are linked via non-covalent bonds to form a quaternary structure.

[0027] The term "pair of affinity domains" refers to 1 × 10 -7 Less than M (e.g., 1 × 10 -8 Less than M, 1 x 10-9 Less than M, 1 x 10 -10 Less than M or 1 x 10 -11 K (less than M) D A pair of affinity domains is two different protein domain(s) that specifically bind to each other at a specific site. In some examples, a pair of affinity domains can be a pair of naturally occurring proteins. In some embodiments, a pair of affinity domains can be a pair of synthetic proteins. Non-limiting examples of paired affinity domains are described herein.

[0028] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen-binding domain. An epitope may consist of, for example, surface-accessible amino acid residues and / or sugar side chains, and may have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former may be lost in the presence of denaturing solvents, but the binding to the latter may not be lost. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. Methods for identifying the epitope bound by an antigen-binding domain are known in the art.

[0029] "Immune effector cell" refers to a cell of the immune system of a mammal that can directly or indirectly recognize and / or cause cytostasis or cell death of pathogenic cells (e.g., cancer cells) in a mammal. Non-limiting examples of immune effector cells include macrophages, T lymphocytes (e.g., cytotoxic T lymphocytes and T helper cells), natural killer cells, neutrophils, monocytes, and eosinophils. Additional examples of immune effector cells are known in the art.

[0030] The term "treatment" refers to ameliorating at least one symptom of a disorder. In some examples, the disorder being treated is cancer, and ameliorating at least one symptom of cancer includes reducing abnormal proliferation, gene expression, signal transduction, translation, and / or factor secretion. Generally, treatment methods involve administering to a subject in need of, or determined to be in need of, such treatment, a therapeutically effective amount of a composition that alleviates at least one symptom of the disorder.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, data, and other supporting documents mentioned herein are incorporated by reference. Database entries, and other references are incorporated by reference in their entireties. In the event of a conflict, the present specification, including definitions, will control.

[0032] [The present invention 1001] (a)(i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains; a first chimeric polypeptide comprising: (b)(i) a second domain of the pair of affinity domains; (ii) a second target-binding domain; and a second chimeric polypeptide comprising wherein the first chimeric polypeptide and the second chimeric polypeptide are associated via binding between the first domain and the second domain of the pair of affinity domains. [The present invention 1002] 1001. The multi-chain chimeric polypeptide of the present invention, wherein said first target binding domain and said soluble tissue factor domain are immediately adjacent to each other within said first chimeric polypeptide. [The present invention 1003] 1001. The multi-chain chimeric polypeptide of the present invention, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide. [The present invention 1004] The multi-chain chimeric polypeptide of any of claims 1001 to 1003, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are immediately adjacent to each other within the first chimeric polypeptide. [The present invention 1005] The multi-chain chimeric polypeptide of any of claims 1001 to 1003, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain of the pair of affinity domains. [The present invention 1006] The multi-chain chimeric polypeptide of any of claims 1001 to 1005, wherein the second domain and the second target binding domain of the pair of affinity domains are immediately adjacent to each other within the second chimeric polypeptide. [The present invention 1007] A multi-chain chimeric polypeptide of any of claims 1001 to 1005, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. [The present invention 1008] The multi-chain chimeric polypeptide of any of claims 1001 to 1007, wherein the first target-binding domain and the second target-binding domain specifically bind to the same antigen. [The present invention 1009] The multi-chain chimeric polypeptide of any of claims 1001 to 1007, wherein the first target-binding domain and the second target-binding domain specifically bind to different antigens. [The present invention 1010] The multi-chain chimeric polypeptide of any of claims 1001 to 1009, wherein one or both of said first target-binding domain and said second target-binding domain is an antigen-binding domain. [The present invention 1011] One or both of the first target binding domain and the second target binding domain may be selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, and ULBP2. , CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P -Cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII 10. The multi-chain chimeric polypeptide of any of claims 1001 to 1010, which specifically binds to a target selected from the group consisting of: a TGF-βRIII ligand, a DNAM-1 ligand, an NKp46 ligand, an NKp44 ligand, an NKG2D ligand, an NKp30 ligand, a scMHCI ligand, a scMHCII ligand, a scTCR ligand, an IL-1 receptor, an IL-2 receptor, an IL-3 receptor, an IL-7 receptor, an IL-8 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-17 receptor, an IL-18 receptor, an IL-21 receptor, a PDGF-DD receptor, a stem cell factor (SCF) receptor, a stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, a MICA receptor, a MICB receptor, a ULP16 binding protein receptor, a CD155 receptor, a CD122 receptor, and a CD28 receptor. [The present invention 1012] The multi-chain chimeric polypeptide of any of claims 1001 to 1010, wherein one or both of said first target-binding domain and said second target-binding domain is a soluble interleukin or cytokine protein. [The present invention 1013] The multi-chain chimeric polypeptide of the present invention, wherein the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. [The present invention 1014] The multi-chain chimeric polypeptide of any of claims 1001 to 1010, wherein one or both of said first target-binding domain and said second target-binding domain is a soluble interleukin or cytokine receptor. [The present invention 1015] The soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble N A multichain chimeric polypeptide of the invention 1014 which is Kp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28. [The present invention 1016] 1016. The multi-chain chimeric polypeptide of any of claims 1001 to 1015, wherein said first chimeric polypeptide further comprises one or more additional target binding domain(s). [The present invention 1017] 1017. The multi-chain chimeric polypeptide of any of claims 1001 to 1016, wherein said second chimeric polypeptide further comprises one or more additional target binding domains. [The present invention 1018] The multi-chain chimeric polypeptide of any of claims 1001 to 1017, wherein the soluble tissue factor domain is a soluble human tissue factor domain. [The present invention 1019] 1018. The multi-chain chimeric polypeptide of the invention, wherein said soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:1. [The present invention 1020] The multi-chain chimeric polypeptide of any of claims 1001 to 1019, wherein the pair of affinity domains is a sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. [The present invention 1021] 1001-1019. The multi-chain chimeric polypeptide of any of claims 1001 to 1019, wherein the pair of affinity domains is selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter / docking tag modules based on mutant RNase I fragments, and SNARE modules based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. [The present invention 1022] The multi-chain chimeric polypeptide of any of claims 1001 to 1021, wherein the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus. [The present invention 1023] A composition comprising any one of the multi-chain chimeric polypeptides of the present invention 1001 to 1022. [The present invention 1024] The composition of the present invention 1023, which is a pharmaceutical composition. [The present invention 1025] A kit comprising at least one dose of the composition of the present invention 1023 or 1024. [The present invention 1026] 1. A method for stimulating immune cells, comprising: contacting immune cells with an effective amount of any of the multi-chain chimeric polypeptides of the present invention 1001 to 1022 or the composition of the present invention 1023 or 1024; A method comprising: [The present invention 1027] 1. A method for inducing or increasing immune cell proliferation, comprising: contacting immune cells with an effective amount of any of the multi-chain chimeric polypeptides of the present invention 1001 to 1022 or the composition of the present invention 1023 or 1024; A method comprising: [The present invention 1028] 1. A method for inducing differentiation of immune cells into memory or memory-like immune cells, comprising: contacting immune cells with an effective amount of any of the multi-chain chimeric polypeptides of the present invention 1001 to 1022 or the composition of the present invention 1023 or 1024; A method comprising: [The present invention 1029] The method of any one of claims 1026 to 1028, wherein the immune cells are contacted in vitro. [The present invention 1030] The method of any one of claims 1026 to 1028, wherein the immune cells are contacted in vivo. [The present invention 1031] 1030. The method of any one of claims 1026 to 1030, wherein the immune cells are selected from the group consisting of immature thymocytes, peripheral blood lymphocytes, naive T cells, multipotent Th cell precursors, lymphoid progenitor cells, Treg cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8+ T cells, CD4+ T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells. [The present invention 1032] The method of any of claims 1026 to 1031, wherein the immune cells have been genetically modified in advance to express a chimeric antigen receptor or a recombinant T cell receptor. [The present invention 1033] A method for killing cancer cells, infected cells, or senescent cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of the present inventions 1001 to 1022 or the composition of the present invention 1023 or 1024. [The present invention 1034] A method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of the present inventions 1001 to 1022 or the composition of the present invention 1023 or 1024. [This invention 1035] The method of any one of claims 1033 to 1034, wherein said subject has been identified or diagnosed as having cancer, an age-related disease or condition, or an infectious disease. [The present invention 1036] 1035. The method of claim 1035, wherein said cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. [This invention 1037] 1035. The method of claim 1035, wherein the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. [The present invention 1038] 1035. The method of claim 1035, wherein said infectious disease is infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus. [This invention 1039] A nucleic acid encoding any one of the multi-chain chimeric polypeptides of the present invention 1001 to 1022. [The present invention 1040] A vector comprising the nucleic acid of the present invention. [The present invention 1041] A cell comprising the nucleic acid of the present invention 1039 or the vector of the present invention 1040. [The present invention 1042] 1. A method for producing a multi-chain chimeric polypeptide, comprising: Culturing the cells of invention 1041 in a culture medium under conditions sufficient to result in the production of said multi-chain chimeric polypeptide; recovering said multi-chain chimeric polypeptide from said cells and / or said culture medium. A method comprising: [This invention 1043] A multi-chain chimeric polypeptide produced by the method of the present invention. [This invention 1044] The multi-chain chimeric polypeptide of the present invention, wherein the human soluble tissue factor domain does not initiate blood coagulation. [This invention 1045] The multi-chain chimeric polypeptide of any of claims 1001 to 1022, wherein the soluble tissue factor domain comprises or consists of a sequence derived from wild-type soluble human tissue factor. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [Brief explanation of the drawings]

[0033] [Figure 1]Figure 1 shows an exemplary diagram of a multi-chain chimeric polypeptide: (i) a first chimeric polypeptide comprising a first target-binding domain (A), a soluble tissue factor domain, a first domain of a pair of affinity domains (soluble interleukin IL-15), and an additional target-binding domain (B); and (ii) a second chimeric polypeptide comprising a second domain of a pair of affinity domains (IL-15 receptor alpha sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). The top schematic shows the association of the first chimeric polypeptide with the second chimeric polypeptide via the pair of affinity domains. The bottom schematic shows the order of the domains within the first and second chimeric polypeptides. [Figure 2] Figure 1 shows exemplary diagrams of multi-chain chimeric polypeptides: (i) a first chimeric polypeptide comprising a first target-binding domain (A), a soluble tissue factor domain containing five amino acid substitutions to eliminate binding of the soluble tissue factor domain to FVIIa, a first domain of a pair of affinity domains (soluble interleukin IL-15 containing D8N or D8A amino acid substitutions), and an additional target-binding domain (B); and (ii) a second chimeric polypeptide comprising a second domain of a pair of affinity domains (IL-15 receptor alpha sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). The top schematic shows the association of the first and second chimeric polypeptides via the pair of affinity domains. The bottom schematic shows the order of the domains within the first and second chimeric polypeptides. In other embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain may comprise or consist of a soluble wild-type human tissue factor domain (comprising or consisting of a contiguous sequence within wild-type human tissue factor). [Figure 3] 1 shows a schematic diagram of an exemplary IL-12 / IL-15RαSu DNA construct. [Figure 4] Schematic diagram of an exemplary IL-18 / TF / IL-15 DNA construct is shown. [Figure 5]FIG. 1 shows a schematic diagram of the interaction between exemplary IL-12 / IL-15RαSu and IL-18 / TF / IL-15 DNA constructs. [Figure 6] FIG. 1 shows a schematic diagram of the interaction between an exemplary IL-12 / IL-15RαSu fusion protein and an IL-18 / TF / IL-15 fusion protein, resulting in an IL-18 / TF / IL-15:IL-12 / IL-15RαSu complex (18t15-12s). [Figure 7] Chromatograph of purified 18t15-12s elution from an anti-TF antibody affinity column. [Figure 8] 1 shows an exemplary chromatographic profile of anti-TF antibody / SEC-purified 18t15-12s protein after elution on an analytical size-exclusion column, demonstrating the separation of the monomeric multiprotein 18t15-12s complex from protein aggregates. [Figure 9] An example of 4-12% SDS-PAGE of the 18t15-12s complex after disulfide bond reduction is shown. Lane 1: SeeBlue Plus2 marker, Lane 2: anti-tissue factor antibody affinity column-purified 18t15-12s (0.5 μg), Lane 3: anti-tissue factor antibody affinity column-purified 18t15-12s (1 μg). [Figure 10] SDS PAGE analysis of deglycosylated and non-deglycosylated 18t15-12s. Lane 1: anti-tissue factor antibody affinity column-purified 18t15-12s (0.5 μg) (non-deglycosylated). Lane 2: anti-TF antibody-purified 18t15-12s (1 μg) (non-deglycosylated). Lane 3: 18t15-12s (1 μg) (deglycosylated). Lane 4: Mark12 unstained marker. [Figure 11] Sandwich ELISA of the 18t15-12s complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 detection antibody (BAF 219) is shown. [Figure 12] Sandwich ELISA of the 18t15-12s complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247) is shown. [Figure 13]Sandwich ELISA of the 18t15-12s complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-18 detection antibody (D045-6) is shown. [Figure 14] Sandwich ELISA of the 18t15-12s complex containing an anti-human tissue factor (I43) capture antibody and an anti-human tissue factor detection antibody. [Figure 15] IL-15-dependent 32D β-cell proliferation mediated by the 18t15-12s complex (open squares) and recombinant IL-15 (closed squares) is shown. [Figure 16] The biological activity of IL-18 in the 18t15-12s complex (open squares) is shown, with recombinant IL-18 (closed squares) and recombinant IL-12 (closed circles) serving as positive and negative controls, respectively. [Figure 17] The biological activity of IL-12 in the 18t15-12s complex (open squares) is shown, with recombinant IL-12 (closed circles) and recombinant IL-18 (closed squares) serving as positive and negative controls, respectively. [Figure 18A] 1 shows the cell surface expression of CD25 on NK cells induced by the 18t15-12s complex. [Figure 18B] 1 shows cell surface CD69 expression on NK cells induced by the 18t15-12s complex. [Figure 19] 1 shows a flow cytometry graph of intracellular interferon gamma expression in NK cells induced by the 18t15-12s complex. [Figure 20] 1 shows the cytotoxicity of 18t15-12s-induced human NK cells against K562 cells. [Figure 21] 1 shows a schematic diagram of an exemplary IL-12 / IL-15RαSu / αCD16 DNA construct. [Figure 22] Schematic diagram of an exemplary IL-18 / TF / IL-15 DNA construct is shown. [Figure 23] FIG. 1 shows a schematic diagram of the interaction between an exemplary IL-12 / IL-15RαSu / αCD16scFv DNA construct and an IL-18 / TF / IL-15 DNA construct. [Figure 24] 1 shows a schematic diagram of an exemplary 18t15-12s / αCD16 protein complex. [Figure 25] 1 shows a sandwich ELISA of the 18t15-12s16 complex with an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 or IL-18 detection antibody. [Figure 26] 1 shows a schematic diagram of an exemplary TGFβRII / IL-15RαSu DNA construct. [Figure 27] 1 shows a schematic diagram of an exemplary IL-21 / TF / IL-15 construct. [Figure 28] FIG. 1 shows a schematic diagram of the interaction between an exemplary IL-IL-21 / TF / IL-15 construct and a TGFβRII / IL-15RαSu construct. [Figure 29] FIG. 1 shows a schematic diagram of the interaction between an exemplary TGFβRII / IL-15RαSu fusion protein and an IL-21 / TF / IL-15 fusion protein, resulting in an IL-21 / TF / IL-15 / TGFβRII / IL-15RαSu complex (21t15-TGFRs). [Figure 30] Chromatograph of purified 21t15-TGFRs eluted from an anti-TF antibody affinity column. [Figure 31] 1 shows an exemplary 21t15-TGFRs size exclusion chromatogram showing the main protein peak and a high molecular weight peak. [Figure 32] An example of 4-12% SDS-PAGE of the 21t15-TGFRs complex after disulfide bond reduction is shown. Lane 1: Mark12 unstained marker (numbers on the left indicate molecular weight in kDa), lane 2: 21t15-TGFRs (0.5 μg), lane 3: 21t15-TGFRs (1 μg), lane 4: 21t15-TGFRs (deglycosylated) (1 μg). The expected MW was 53 kDa and 39.08 kDa. [Figure 33] Sandwich ELISA of 21t15-TGFRs complexes containing anti-human tissue factor capture antibody and biotinylated anti-human IL-21 detection antibody (13-7218-81, BioLegend) is shown. [Figure 34]Sandwich ELISA of 21t15-TGFRs complexes containing anti-human tissue factor capture antibody and biotinylated anti-human IL-15 detection antibody (BAM 247, R&D Systems) is shown. [Figure 35] Sandwich ELISA of 21t15-TGFRs complexes containing anti-human tissue factor capture antibody and biotinylated anti-human TGFβRII detection antibody (BAF241, R&D Systems) is shown. [Figure 36] Sandwich ELISA of 21t15-TGFRs complexes containing anti-human tissue factor (I43) capture antibody and anti-human tissue factor detection antibody. [Figure 37] IL-15 dependent proliferation of 32D β cells mediated by 21t15-TGFRs complex (open squares) compared to IL-15 (closed squares). [Figure 38] The biological activity of the TGFβRII domain within the 21t15-TGFRs complex (open squares) is shown. TGFβRII / Fc (closed squares) served as a positive control. [Figure 39] 1 shows a flow cytometry graph of cell surface CD25 expression of NK cells induced by the 21t15-TGFRs complex. [Figure 40] 1 shows a flow cytometry graph of cell surface CD69 expression of NK cells induced by the 21t15-TGFRs complex. [Figure 41] 1 shows a flow cytometry graph of intracellular interferon gamma expression in NK cells induced by the 21t15-TGFRs complex. [Figure 42] 1 shows the cytotoxicity of 21t15-TGFRs-induced human NK cells against K562 cells. [Figure 43] Schematic diagram of an exemplary IL-7 / IL-15RαSu DNA construct is shown. [Figure 44] Schematic diagram of an exemplary IL-21 / TF / IL-15 DNA construct is shown. [Figure 45] FIG. 1 shows a schematic diagram of the interaction between an exemplary IL-7 / IL-15RαSu construct and an IL-21 / TF / IL-15 DNA construct. [Figure 46] FIG. 1 shows a schematic diagram of the interaction between an exemplary IL-7 / IL-15RαSu fusion protein and an IL-21 / TF / IL-15 fusion protein, resulting in an IL-21 / TF / IL-15:IL-7 / IL-15RαSu complex (21t15-7s). [Figure 47] This shows the proliferation of primary natural killer (NK) cells stimulated with 21t15-7s+anti-TF IgG1 antibody. [Figure 48] Activation of expanded primary NK cells is shown, using CD25 MFI and CD69 MFI as markers of NK cell activation. [Figure 49] The cytotoxic activity of expanded NK cells against K562 human tumor cells is shown, and NK cells stimulated with 21t15-7s + anti-TF IgG1 antibody show higher specific lysis of K562 cells than NK cells not stimulated with 21t15-7s + anti-TF IgG1 antibody. [Figure 50] 1 shows a schematic diagram of an exemplary IL-21 / IL-15RαSu DNA construct. [Figure 51] Schematic diagram of an exemplary IL-7 / TF / IL-15 DNA construct is shown. [Figure 52] FIG. 1 shows a schematic diagram of the interaction between exemplary IL-21 / IL-15RαSu and IL-7 / TF / IL-15 DNA constructs. [Figure 53] FIG. 1 shows a schematic diagram of the interaction between an exemplary IL-21 / IL-15RαSu fusion protein and an IL-7 / TF / IL-15 fusion protein, resulting in an IL-7 / TF / IL-15:IL-21 / IL-15RαSU complex (7t15-21s). [Figure 54] Activation and proliferation of primary natural killer (NK) cells upon stimulation with 21t15-TGFRs plus anti-TF IgG1 antibody are shown in graphic form. [Figure 55] 1 shows size exclusion chromatography (SEC) profiles of anti-TF IgG1 antibody, 7t15-21s, and a complex containing equal amounts of anti-TF IgG1 antibody and 7t15-21s. [Figure 56]Shown is the oxygen consumption rate (OCR) in picomoles / min of human NK cells (2 x 106 cells / mL) isolated from the blood of two different donors. [Figure 57] Figure 1 shows the extracellular acidification rate (ECAR) in mpH / min of human NK cells (2 x 106 cells / mL) isolated from the blood of two different donors. [Figure 58] A schematic diagram of the 7t15-16s21 construct is shown. [Figure 59] An additional schematic of the 7t15-16s21 construct is shown. [Figure 60] Figures 60A and 60B show the binding of 7t15-16s21 to CHO cells expressing human CD16b compared to a control protein. [Figure 61A] These are the results of an ELISA experiment using an antibody against IL-15 to detect 7t15-16s21. [Figure 61B] This shows the results of an ELISA experiment using an antibody against IL-21 to detect 7t15-16s21. [Figure 61C] This shows the results of an ELISA experiment using an antibody against IL-7 to detect 7t15-16s21. [Figure 62] 1 shows the results of a 32D β cell proliferation assay using 7t15-16s21 or recombinant IL-15. [Figure 63] Chromatographic profile of 7t15-16s21 protein-containing cell culture supernatant after binding to and elution from anti-TF antibody resin. [Figure 64] 1 shows the analytical SEC profile of 7t15-16s21. [Figure 65] A schematic diagram of the TGFRt15-16s21 construct is shown. [Figure 66] An additional schematic of the TGFRt15-16s21 construct is shown. [Figure 67]Figures 67A and 67B show the binding affinity of TGFRt15-16S21 and 7t15-21s to CHO cells expressing human CD16b. Figure 67A shows the binding affinity of TGFRt15-16S21 to CHO cells expressing human CD16b. Figure 67B shows the binding affinity of 7t15-21s to CHO cells expressing human CD16b. [Figure 68] The results of TGFβ1 inhibition by TGFRt15-16s21 and TGFR-Fc are shown. [Figure 69] 1 shows the results of a 32D β cell proliferation assay using TGFRt15-16s21 or recombinant IL-15. [Figure 70A] The results of ELISA to detect IL-15 in TGFRt15-16s21 with the corresponding antibody are shown. [Figure 70B] The results of ELISA to detect IL-21 in TGFRt15-16s21 with the corresponding antibody are shown. [Figure 70C] The results of detecting TGFβRII in TGFRt15-16s21 with the corresponding antibody using ELISA are shown. [Figure 71] 1 shows the chromatographic profile of TGFRt15-16s21 protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody resin. [Figure 72] The results of reducing SDS-PAGE analysis of TGFRt15-16s21 are shown. [Figure 73] A schematic diagram of the 7t15-7s construct is shown. [Figure 74] An additional schematic of the 7t15-7s construct is shown. [Figure 75] Chromatographic profile of 7t15-7s protein-containing cell culture supernatant after binding to and elution from anti-TF antibody resin. [Figure 76] 1 shows detection of TF, IL-15, and IL-7 in 7t15-7s using ELISA. [Figure 77] A schematic diagram of the TGFRt15-TGFRs construct is shown. [Figure 78]An additional schematic of the TGFRt15-TGFRs construct is shown. [Figure 79] The results of TGFβ1 inhibition by TGFRt15-TGFRs and TGFR-Fc are shown. [Figure 80] 1 shows the results of a 32D β cell proliferation assay using TGFRt15-TGFRs or recombinant IL-15. [Figure 81] Figures 81A and 81B show the results of detecting IL-15 and TGFβRII in TGFRt15-TGFRs with the corresponding antibodies using ELISA. [Figure 82] 1 is a line graph showing the chromatographic profile of TGFRt15-TGFRs protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody resin. [Figure 83] 1 shows the analytical SEC profile of TGFRt15-TGFRs. [Figure 84] Shown are TGFRt15-TGFRs before and after deglycosylation analyzed by reducing SDS-PAGE. [Figure 85A] Figures 85A and 85B show spleen weights and percentages of immune cell types in TGFRt15-TGFRs-treated and control-treated mice. Figure 85A shows spleen weights in mice treated with TGFRt15-TGFRs compared to PBS controls. Figure 85B shows the percentages of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with TGFRt15-TGFRs compared to PBS controls. [Figure 85B] See the description of Figure 85A. [Figure 86A] Figures 86A and 86B show spleen weight and immune stimulation over 92 hours in mice treated with TGFRt15-TGFRs. Figure 86A shows the spleen weight of mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figure 86B shows the percentage of immune cells in mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. [Figure 86B] See the description of Figure 86A. [Figure 87A] Ki67 expression over time in mice treated with TGFRt15-TGFRs. [Figure 87B] Granzyme B expression over time in mice treated with TGFRt15-TGFRs. [Figure 88] 1 shows enhanced cytotoxicity of splenocytes by TGFRt15-TGFRs in C57BL / 6 mice. [Figure 89] 1 shows the change in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or the combination of chemotherapy and TGFRt15-TGFRs in a mouse model of pancreatic cancer. [Figure 90] 1 shows the cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFRs. [Figure 91] A schematic diagram of the 7t15-21s137L (long version) construct is shown. [Figure 92] An additional schematic of the 7t15-21s137L (long version) construct is shown. [Figure 93] 1 is a line graph showing the chromatographic profile of 7t15-21s137L (long version) protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody resin. [Figure 94] The analytical SEC profile of 7t15-21s137L (long version) is shown. [Figure 95] Binding of 7t15-21s137L (short version) to CD137L (4.1BBL) is shown. [Figure 96A]Figures 96A-96C show the detection of IL15, IL21, and IL7 in 7t15-21s137L (short version) using ELISA. Figure 96A shows the detection of IL15 in 7t15-21s137L (short version) using ELISA. Figure 96B shows the detection of IL21 in 7t15-21s137L (short version) using ELISA. Figure 96C shows the detection of IL7 in 7t15-21s137L (short version) using ELISA. [Figure 96B] See the description of Figure 96A. [Figure 96C] See the description of Figure 96A. [Figure 97] 1 shows the results of a CTLL-2 cell proliferation assay. [Figure 98] 1 shows the activity of 7t15-1s137L (short version) in promoting proliferation of IL21R-containing B9 cells. [Figure 99] A schematic diagram of the 7t15-TGFRs construct is shown. [Figure 100] An additional schematic of the 7t15-TGFRs construct is shown. [Figure 101] The results of TGFβ1 inhibition by 7t15-TGFRs and TGFR-Fc are shown. [Figure 102A] 102A-102C show the detection of IL-15, TGFβRII, and IL-7 in 7t15-TGFRs using ELISA. [Figure 102B] See the description of Figure 102A. [Figure 102C] See the description of Figure 102A. [Figure 103] 1 shows the results of a 32D β cell proliferation assay using 7t15-TGFRs or recombinant IL-15. [Figure 104] 1 is a line graph showing the chromatographic profile of 7t15-TGFRs protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody affinity column. [Figure 105]Shown are 7t15-TGFRs before and after deglycosylation analyzed using reducing SDS-PAGE. [Figure 106] ELISA detection of IL-7, IL-15, and TGFβRII in 7t15-TGFRs protein. [Figure 107] Figures 107A and 107B show spleen weights and percentages of immune cell types in 7t15-TGFRs-treated and control-treated mice. Figure 107A shows the spleen weights of mice treated with various doses of 7t15-TGFRs compared to the PBS control. Figure 107B shows the percentages of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with various doses of 7t15-TGFRs compared to the PBS control. [Figure 108A] 1 shows upregulation of CD44 expression on CD4+ T cells by 7t15-TGFRs in C57BL / 6 mice. [Figure 108B] 1 shows upregulation of CD44 expression on CD8+ T cells by 7t15-TGFRs in C57BL / 6 mice. [Figure 109A] 1 shows upregulation of Ki67 expression in CD8+ T cells and NK cells by 7t15-TGFRs in C57BL / 6 mice. [Figure 109B] 1 shows upregulation of granzyme B expression in CD8+ T cells and NK cells by 7t15-TGFRs in C57BL / 6 mice. [Figure 110] 1 shows the enhanced cytotoxicity of splenocytes by 7t15-TGFRs in C57BL / 6 mice. [Figure 111] A schematic diagram of the TGFRt15-21s137L construct is shown. [Figure 112] An additional schematic of the TGFRt15-21s137L construct is shown. [Figure 113] 1 is a line graph showing the chromatographic profile of TGFRt15-21s137L protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody affinity column. [Figure 114] A schematic diagram of the TGFRt15-TGFRs21 construct is shown. [Figure 115] An additional schematic of the TGFRt15-TGFRs21 construct is shown. [Figure 116] 1 is a line graph showing the chromatographic profile of TGFRt15-TGFRs21 protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody affinity column. [Figure 117] Shown is TGFRt15-TGFRs21 before and after deglycosylation analyzed by reducing SDS-PAGE. [Figure 118] Figures 118A and 118B show the detection of the TGFRt15-TGFRs21 component using ELISA. [Figure 119] Figures 119A and 119B show the percentage and proliferation of CD4+ T cells, CD8+ T cells, and natural killer (NK) cells present in the spleens of control-treated and TGFRt15-TGFRs21-treated mice. [Figure 120] 1 shows upregulation of granzyme B expression in splenocytes in mice treated with TGFRt15-TGFRs21. [Figure 121] 1 shows the enhanced cytotoxicity of splenocytes by TGFRt15-TGFRs21 in C57BL / 6 mice. [Figure 122] A schematic diagram of the TGFRt15-TGFRs16 construct is shown. [Figure 123] An additional schematic of the TGFRt15-TGFRs16 construct is shown. [Figure 124] A schematic diagram of the TGFRt15-TGFRs137L construct is shown. [Figure 125] An additional schematic of the TGFRt15-TGFRs137L construct is shown. [Figure 126] 1 shows changes in the surface phenotype of lymphocyte populations following stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s. [Figure 127]1 shows an increase in phospho-STAT4 and phospho-STAT5 levels in NK cells after stimulation with 18t15-12s. [Figure 128A] 128A-128C show in vivo stimulation of Treg cells, NK cells, and CD8+ T cells in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 128B] See the description of Figure 128A. [Figure 128C] See the description of Figure 128A. [Figure 129A] Figures 129A-C show immune stimulation in C57BL / 6 mice after treatment with TGFRt15-TGFRs. [Figure 129B] See the description of Figure 129A. [Figure 129C] See the description of Figure 129A. [Figure 130] Figures 130A and 130B show the in vivo induction of NK cell and CD8+ T cell proliferation in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 131] Figures 131A and 131B show the enhancement of NK cell cytotoxicity after treating NK cells with TGFRt15-TGFRs. [Figure 132] Figures 132A and 132B show the enhancement of ADCC activity of NK cells after treatment of NK cells with TGFRt15-TGFRs. [Figure 133A] Figures 133A-133H show the anti-tumor activity of TGFRt15-TGFRs + anti-TRP1 antibody (TA99) in combination with chemotherapy in a melanoma mouse model. [Figure 133B] See the description of Figure 133A. [Figure 133C] See the description of Figure 133A. [Figure 133D] See the description of Figure 133A. [Figure 133E] See the description of Figure 133A. [Figure 133F]See the description of Figure 133A. [Figure 133G] See the description of Figure 133A. [Figure 133H] See the description of Figure 133A. [Figure 134] Figures 134A-134C show amelioration of Western diet-induced hyperglycemia in ApoE- / - mice by TGFRt15-TGFRs. [Figure 135] Cell surface staining summarizing the differentiation of NK cells into cytokine-induced memory-like NK cells (CIML-NK cells) after stimulation with 18t15-12s and culture in rhIL15 is shown. [Figure 136] Figure 1 shows upregulation of CD44hi memory T cells upon treatment with TGFRt15-TGFRs. [Figure 137] 1 shows a graph of Factor X (FX) activation after treatment with single-chain or multi-chain chimeric polypeptides. [Figure 138] 1 shows the clotting time of buffer solutions with various concentrations of Innovin in a prothrombin time (PT) test. [Figure 139] 1 shows the clotting time of multi-chain chimeric polypeptides in a PT assay. [Figure 140] 1 shows the clotting time of multi-chain chimeric polypeptides in a PT assay when mixed with 32DB cells. [Figure 141] 1 shows the clotting time of multi-chain chimeric polypeptides in a PT assay when mixed with human PBMCs. [Figure 142] Binding of 7t15-21s137L (long version) and 7t15-21s137L (short version) to CD137 (4.1BB) is shown. [Figure 143A] Figures 143A to 143D show the detection of IL7, IL21, IL15, and 4.1BBL in 7t15-21s137L (long version) by their respective antibodies using ELISA. [Figure 143B] See the description of Figure 143A. [Figure 143C]See the description of Figure 143A. [Figure 143D] See the description of Figure 143A. [Figure 144] IL-15 activity of 7t15-21s137L (long version) and 7t15-21s137L (short version) assessed by IL2Rαβγ-containing CTLL2 cell proliferation assay is shown. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description Provided herein is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising (i) a first target-binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising (i) a second domain of the pair of affinity domains and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated via the binding between the first domain and the second domain of the pair of affinity domains. Also provided herein are compositions comprising any of the multi-chain chimeric polypeptides described herein, nucleic acids encoding any of the multi-chain chimeric polypeptides described herein, and cells comprising any of the nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods of stimulating immune cells and methods of treating a subject in need of treatment, comprising the use of any of the multi-chain chimeric polypeptides described herein. Also provided herein are methods of producing any of the multi-chain chimeric polypeptides described herein.

[0035] In some examples of any of the multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide is, independently, from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, from about 50 amino acids to about 700 amino acids, from about 50 amino acids to about 650 amino acids, from about 50 amino acids to about 600 amino acids, from about 50 amino acids to about 550 amino acids, from about 50 amino acids to about 500 amino acids, from about 50 amino acids to about 480 amino acids, from about 50 amino acids to about 460 amino acids, from about 50 amino acids to about 440 amino acids, from about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, About 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 5 00 amino acids, about 100 to about 480 amino acids, about 100 to about 460 amino acids, about 100 to about 440 amino acids, about 100 to about 420 amino acids, about 100 to about 400 amino acids, about 100 to about 380 amino acids, about 100 to about 360 amino acids, about 100 to about 340 amino acids, about 100 to about 320 amino acids, about 100 to about 300 amino acids, about 100 to about 280 amino acids, about 100 to about 260 amino acids, about 100 to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids,About 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids amino acids, about 150 to about 280 amino acids, about 150 to about 260 amino acids, about 150 to about 240 amino acids, about 150 to about 220 amino acids, about 150 to about 200 amino acids, about 200 to about 3000 amino acids, about 200 to about 2500 amino acids, about 200 to about 2000 amino acids, about 200 to about 1500 amino acids, about 200 to about 1000 amino acids, about 200 to about 950 amino acids, about 200 to about 900 amino acids, about 200 to about 850 amino acids, about 200 to about About 800 amino acids, about 200 to about 750 amino acids, about 200 to about 700 amino acids, about 200 to about 650 amino acids, about 200 to about 600 amino acids, about 200 to about 550 amino acids, about 200 to about 500 amino acids, about 200 to about 480 amino acids, about 200 to about 460 amino acids, about 200 to about 440 amino acids, about 200 to about 420 amino acids, about 200 to about 400 amino acids, about 200 to about 380 amino acids, about 200 to about 360 amino acids, about 200 amino acids amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids,Approximately 220A, amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 9 00 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids,About 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about About 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids,Approximately 300A, amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids, about 320 amino acids to about 340 amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, About 340 amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids , about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3 000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids,about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, About 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 400 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 48 ...500 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 20 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids,About 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, About 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids,Approximately 600 to 1000 amino acids, approximately 600 to 950 amino acids, About 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 8 50 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 Amino acids up to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1 000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids,The length may be about 900 to about 1500 amino acids, about 900 to about 1000 amino acids, about 900 to about 950 amino acids, about 950 to about 3000 amino acids, about 950 to about 2500 amino acids, about 950 to about 2000 amino acids, about 950 to about 1500 amino acids, about 950 to about 1000 amino acids, about 1000 to about 3000 amino acids, about 1000 to about 2500 amino acids, about 1000 to about 2000 amino acids, about 1000 to about 1500 amino acids, about 1500 to about 3000 amino acids, about 1500 to about 2500 amino acids, about 1500 to about 2000 amino acids, about 2000 to about 3000 amino acids, about 2000 to about 2500 amino acids, or about 2500 to about 3000 amino acids. Diagrams of exemplary multi-chain chimeric polypeptides provided herein are shown in Figures 1 and 2.

[0036] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are immediately adjacent to each other within the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) within the first chimeric polypeptide.

[0037] In some embodiments of any of the multi-chain chimeric polypeptides described herein, a soluble tissue factor domain (e.g., an exemplary soluble tissue factor domain described herein) is used. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the paired affinity domain (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide.

[0038] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) in the second chimeric polypeptide.

[0039] Non-limiting aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are described below and may be used in any combination without limitation. Additional aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are known in the art.

[0040] tissue factor Human tissue factor is a 263-amino acid transmembrane protein containing three domains: (1) a 219-amino acid N-terminal extracellular domain (residues 1–219), (2) a 22-amino acid transmembrane domain (residues 220–242), and (3) a 21-amino acid cytoplasmic C-terminal tail (residues 242–263) (UniProtKB Identifier Number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258 and one S-palmitoylation site at Cys245. Deletions or mutations in the cytoplasmic domain have not been found to affect tissue factor clotting activity. Tissue factor has one S-palmitoylation site within the intracellular domain of the protein at Cys245. Cys245 is located at the amino acid terminus of the intracellular domain, near the membrane surface. The tissue factor transmembrane domain is composed of a single transmembrane α-helix.

[0041] The extracellular domain of tissue factor, consisting of two fibronectin type III domains, is linked to the transmembrane domain via a six-amino acid linker. This linker provides conformational flexibility to separate the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping β-sheets, with the upper sheet domain containing three antiparallel β-strands and the lower sheet containing four β-strands. The β-strands are connected by β-loops between strands βA and βB, βC and βD, and βE and βF, all of which conformations are conserved within the two modules. Three short α-helical segments connect the β-strands. A unique feature of tissue factor is the β-strands. The 17-amino acid β-hairpin between strands β10 and β11 is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is absent in the C-terminal domain and is unique to tissue factor. This fibronectin type III domain structure is characteristic of the immunoglobulin-like protein family fold and is conserved among a wide variety of extracellular proteins.

[0042] Zymogen FVII is rapidly converted to FVIIa by limited proteolysis upon binding to tissues to form the active tissue factor-FVIIa complex. FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa on the tissue factor-FVIIa complex significantly increases the enzymatic activity of FVIIa, increasing the hydrolysis rate of small chromogenic peptidyl substrates by approximately 20- to 100-fold and the activation rate of the natural polymer substrates FIX and FX by nearly one million-fold. In concert with the allosteric activation of the FVIIa active site upon binding to tissue factor, the formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface) induces Ca + ... 2+ It further increases the activation rate of FIX or FX by 1,000-fold in a tissue factor-dependent manner. The approximately 1,000,000-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complexes compared to free FVIIa is a critical regulatory point in the coagulation cascade.

[0043] FVII is a single polypeptide chain of approximately 50 kDa, consisting of 406 amino acid residues, with an N-terminal gamma-carboxyglutamic acid-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EGFR2), and a C-terminal serine protease domain. FVII is characterized by an Ile-Ile linker region in the short linker region between the EGF2 and protease domains. 154 -Arg 152Activation to FVIIa occurs by specific proteolytic cleavage of the bond between the light and heavy chains. 135 and Cys 262 FVIIa binds to Ca via its N-terminal GLA domain. 2+ Immediately C-terminal to the GLA domain is an aromatic stack and two EGF domains. The aromatic stack binds the GLA domain to a single Ca 2+ This Ca 2+ Occupation of the binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consists of His 193 , Asp 242 , and Ser 344 and a single Ca domain in the FVIIa protease domain. 2+ Ion binding is essential for its catalytic activity. The proteolytic activity of FVII to FVIIa is mediated by Ile 153 releases the newly formed amino terminus, folds back, and inserts into the activation pocket, forming Asp 343 It forms a salt bridge with the carboxylate of FVIIa to generate an oxyanion hole. This salt bridge formation is essential for FVIIa activity. However, the formation of the oxyanion hole does not occur in free FVIIa upon proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

[0044] Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is crucial for FVIIa relative to its cognate substrate. Free FVIIa adopts a stable extended conformation upon binding to membranes, with its active site positioned approximately 80 Å above the membrane surface. Upon FVIIa binding to tissue factor, the FVIIa active site is repositioned approximately 6 Å closer to the membrane. This adjustment may aid in the proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLA domain-less FVIIa, it was shown that the active site was still positioned at a similar distance from the membrane, indicating that tissue factor can fully support FVIIa active site positioning even in the absence of FVIIa-membrane interactions. Additional data suggest that tissue factor can bind the intact FVIIa protein as long as the tissue factor extracellular domain is somehow tethered to the membrane surface. However, elevating the FVIIa active site 80 Å above the membrane surface significantly reduced the ability of the tissue factor-FVIIa complex to activate FX, but not the tissue factor-FVIIa amidolytic activity.

[0045] Alanine scanning mutagenesis has been used to evaluate the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitutions identified a limited number of residue positions where alanine substitutions caused a 5- to 10-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be fully exposed to solvent in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand-binding site is located across an extensive region at the interface between the two modules. In the C-module, residues Arg, located on the protruding BC loop, 135 and Phe 140 provides independent contact with FVIIa. 133is located at the base of the finger-like structure and is packed into the gap between the two modules. 20 , Thr 60 , Asp 58 , and Ile 22 Continuity is provided for the major cluster of important binding residues consisting of Thr 60 is only partially solvent exposed and may play a local structural role rather than making significant contact with the ligand. 24 and Gln 110 , as well as potentially more distal residue Val 207 The binding region extends from Asp58 to Lys 48 , Lys 46 , Gln 37 , Asp 44 , and Trp 45 The convex area formed by the Trp 45 and Asp 44 does not interact independently with FVIIa, and Trp 45 The effect of mutation at position 1 is similar to that of the adjacent Asp 44 and Gln 37 The interaction region is located between two surface-exposed aromatic residues, Phe, which form part of a hydrophobic cluster within the N-module. 76 and Tyr 78 Further includes:

[0046] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as certain proteinase-activated receptors. Mutational analysis has identified several residues that, when mutated, support full FVIIa amidolytic activity toward small peptidyl substrates but lack the ability to support activation of macromolecular substrates (i.e., FVII, FIX, and FX) (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9):6375-6381, 1992; Huang et al., J Biol Chem 271(36):21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region from residues 159 to 165, and residues within or adjacent to this flexible loop, have been shown to be essential for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate-binding exosite region of tissue factor, located far from the FVIIa active site. Substitution of the glycine residue with a slightly more bulky alanine residue significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility provided by glycine is essential for the loop from residues 159 to 165 for tissue factor macromolecular substrate recognition.

[0047] Residue Lys 165 and Lys 166 It has also been demonstrated that Lys is important for substrate recognition and binding. Mutation of either of these residues to alanine significantly reduces tissue factor cofactor function. 165 and Lys 166 are facing outwards, and Lys 165 refers to FVIIa in most tissue factor-FVIIa structures, and Lys 166 refers to the substrate-binding exosite region in the crystal structure. Lys of FVIIa 165 and FVIIa Gla 35 Putative salt bridge formation between the GLA domain of tissue factor FVIIa These results suggest that the C-terminal portion of the tissue factor ectodomain interacts directly with the GLA domains of FIX and FX, and with the possible adjacent EGF1 domain, and that the presence of the FVIIa GLA domain may modulate these interactions either directly or indirectly.

[0048] Soluble tissue factor domain In some embodiments of any of the polypeptides, compositions, or methods described herein, the soluble tissue factor domain can be a wild-type tissue factor polypeptide lacking the signal sequence, transmembrane domain, and intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor mutant, where the wild-type tissue factor polypeptide lacks the signal sequence, transmembrane domain, and intracellular domain and is further modified with selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, soluble mouse tissue factor domains, soluble rat tissue factor domains, and mutant soluble tissue factor domains are provided below.

[0049] Exemplary soluble human tissue factor domain (SEQ ID NO: 1) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary nucleic acids encoding soluble human tissue factor domains (SEQ ID NO:2) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGAC AGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 3) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 4) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary soluble mouse tissue factor domain (SEQ ID NO: 5) agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnap kflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge Exemplary soluble rat tissue factor domain (SEQ ID NO: 6) Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

[0050] In some embodiments, the soluble tissue factor domain can comprise a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:1, 3, 4, 5, or 6. In some embodiments, the soluble tissue factor domain may comprise the sequence of SEQ ID NO: 1, 3, 4, 5, or 6 with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its N-terminus and / or 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its C-terminus.

[0051] As can be understood in the art, one of skill in the art will understand that mutations in amino acids that are conserved among different mammalian species are likely to reduce the activity and / or structural stability of a protein, while mutations in amino acids that are not conserved among different mammalian species are unlikely to reduce the activity and / or structural stability of a protein.

[0052] In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain cannot bind to factor VIIa. In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

[0053] In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain. It may be a factor domain.

[0054] In some examples, the soluble tissue factor domain does not include one or more (e.g., 2, 3, 4, 5, 6, or 7) of: a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein, an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein, a tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, an arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and a phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the mutant soluble tissue factor has the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0055] In some examples, the soluble tissue factor domain can be encoded by a nucleic acid comprising a sequence at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:2.

[0056] In some embodiments, the soluble tissue factor domain has a total length of about 20 to about 220 amino acids, about 20 to about 215 amino acids, about 20 to about 210 amino acids, about 20 to about 205 amino acids, about 20 to about 200 amino acids, about 20 to about 195 amino acids, about 20 to about 190 amino acids, about 20 to about 185 amino acids, about 20 to about 180 amino acids, about 20 to about 175 amino acids, about 20 to about 170 amino acids, about 20 to about 165 amino acids, or about 20 to about 160 amino acids. amino acids, about 20 to about 155 amino acids, about 20 to about 150 amino acids, about 20 to about 145 amino acids, about 20 to about 140 amino acids, about 20 to about 135 amino acids, about 20 to about 130 amino acids, about 20 to about 125 amino acids, about 20 to about 120 amino acids, about 20 to about 115 amino acids, about 20 to about 110 amino acids, about 20 to about 105 amino acids, about 20 to about 100 amino acids, about 20 to about 95 amino acids, about 20 to about 90 amino acids, about 20 to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 20 about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids,about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 5, 0 amino acids, about 30 amino acids to about 40 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 215 amino acids, about 40 amino acids to about 210 amino acids, about 40 amino acids to about 205 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 1 to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 215 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 205 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 ... to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids,about 50 to about 95 amino acids, about 50 to about 90 amino acids, about 50 to about 85 amino acids, about 50 to about 80 amino acids, about 50 to about 75 amino acids, about 50 to about 70 amino acids, about 50 to about 60 amino acids, about 60 to about 220 amino acids, about 60 to about 215 amino acids, about 60 to about 210 amino acids, about 60 to about 205 amino acids, about 60 to about 200 amino acids, about 60 to about 195 amino acids, about 60 to about 190 amino acids, about 60 to about 185 amino acids, about 6 0 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids amino acids, about 60 to about 105 amino acids, about 60 to about 100 amino acids, about 60 to about 95 amino acids, about 60 to about 90 amino acids, about 60 to about 85 amino acids, about 60 to about 80 amino acids, about 60 to about 75 amino acids, about 60 to about 70 amino acids, about 70 to about 220 amino acids, about 70 to about 215 amino acids, about 70 to about 210 amino acids, about 70 to about 205 amino acids, about 70 to about 200 amino acids, about 70 to about 195 amino acids, about 70 to about 190 amino acids amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids about 80 to about 185 amino acids, about 80 to about 180 amino acids, about 80 to about 175 amino acids, about 80 to about 170 amino acids, about 80 to about 165 amino acids, about 80 to about 160 amino acids, about 80 to about 155 amino acids, about 80 to about 150 amino acids, about 80 to about 145 amino acids, about 80 to about 140 amino acids, about 80 to about 135 amino acids, about 80 to about 130 amino acids, about 80 to about 125 amino acids, about 80 to about 120 amino acids, about 80 to about 115 amino acids amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 18 ... about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids,about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 1 ...10 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 11 About 210 amino acids, about 110 to about 205 amino acids, about 110 to about 200 amino acids, about 110 to about 195 amino acids, about 110 to about 190 amino acids, about 110 to about 185 amino acids, about 110 to about 180 amino acids, about 110 to about 175 amino acids, about 110 to about 170 amino acids, about 110 to about 165 amino acids, about 110 to about 160 amino acids, about 110 to about 155 amino acids, about 110 to about 150 amino acids, about 110 to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, About 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 Amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 165 amino acids about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 Amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids,About 130 amino acids to about 210 amino acids, about 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 Amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 17 ... about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 Amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids,About 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, About 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 Amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 ... about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 Amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids, about 165 amino acids to about 205 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids,About 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 Amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 215 amino acids, about 180 amino acids to about 210 amino acids, about 180 amino acids to about 205 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 185 amino acids About 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 215 amino acids, about 185 amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids, about 190 amino acids to about 200 amino acids, about 190 Amino acids to about 195 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195 amino acids to about 205 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215 amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205 amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids, about 210 amino acids to about 220 amino acids,about 210 amino acids to about 215 amino acids, or about 215, It may have from about 220 amino acids.

[0057] Linker sequence In some embodiments, the linker sequence may be a flexible linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et al., Protein Engineering, Design & Selection 27(10):325-330, 2014, and Priyanka et al., Protein Sci. 22(2):153-167, 2013. In some examples, the linker sequence is a synthetic linker sequence.

[0058] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequence(s) (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequence(s) (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

[0059] In some embodiments, the linker sequence may have a total length of 1 to about 100 amino acids, 1 to about 90 amino acids, 1 to about 80 amino acids, 1 to about 70 amino acids, 1 to about 60 amino acids, 1 to about 50 amino acids, 1 to about 45 amino acids, 1 to about 40 amino acids, 1 to about 35 amino acids, 1 to about 30 amino acids, 1 to about 25 amino acids, 1 to about 24 amino acids, 1 to about 22 amino acids, 1 to about 20 amino acids, 1 to about 18 amino acids, 1 to about 16 amino acids, 1 to about 20 amino acids, 1 to about 25 amino acids, 1 to about 24 amino acids, 1 to about 22 amino acids, 1 to about 20 amino acids, 1 to about 18 amino acids, 1 to about 16 amino acids, 1 to about 25 ... to about 14 amino acids, 1 to about 12 amino acids, 1 to about 10 amino acids, 1 to about 8 amino acids, 1 to about 6 amino acids, 1 to about 4 amino acids, about 2 to about 100 amino acids, about 2 to about 90 amino acids, about 2 to about 80 amino acids, about 2 to about 70 amino acids, about 2 to about 60 amino acids, about 2 to about 50 amino acids, about 2 to about 45 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 amino acids about 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to about 6 ... amino acids to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 90 amino acids,About 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids, amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 amino acids, about 8 amino acids to about 30 amino acids, about 8 amino acids to about 25 amino acids, about 8 amino acids to about 24 amino acids acid, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids, about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids acid, about 10 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 24 amino acids, about 10 to about 22 amino acids, about 10 to about 20 amino acids, about 10 to about 18 amino acids, about 10 to about 16 amino acids, about 10 to about 14 amino acids, about 10 to about 12 amino acids, about 12 to about 100 amino acids, about 12 to about 90 amino acids, about 12 to about 80 amino acids, about 12 to about 70 amino acids, about 12 to about 60 amino acids, about 12 to about 50 amino acids, about 12 amino acids about 45 amino acids, about 12 amino acids to about 40 amino acids, about 12 amino acids to about 35 amino acids, about 12 amino acids to about 30 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14 amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids, about 14 amino acids to about 60 amino acids,About 14 amino acids to about 50 amino acids, about 14 amino acids to about 45 amino acids, about 14 amino acids to about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, about 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 1 8 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 100 amino acids, about 22 amino acids to about 90 amino acids, about 22 amino acids to about 80 amino acids, about 22 amino acids to about 70 amino acids, about 22 amino acids to about 60 amino acids, about 22 amino acids to about 50 amino acids, about 22 amino acids to about 45 amino acids, about 22 amino acids to about 40 amino acids, about 22 amino acids to about 35 amino acids, about 22 amino acids, amino acids to about 30 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids amino acids, about 30 to about 80 amino acids, about 30 to about 70 amino acids, about 30 to about 60 amino acids, about 30 to about 50 amino acids, about 30 to about 45 amino acids, about 30 to about 40 amino acids, about 30 to about 35 amino acids, about 35 to about 100 amino acids, about 35 to about 90 amino acids, about 35 to about 80 amino acids, about 35 to about 70 amino acids, about 35 to about 60 amino acids, about 35 to about 50 amino acids, about 35 to about 45 amino acids, about 35 to about About 40 amino acids, about 40 to about 100 amino acids, about 40 to about 90 amino acids, about 40 to about 80 amino acids, about 40 to about 70 amino acids, about 40 to about 60 amino acids, about 40 to about 50 amino acids, about 40 to about 45 amino acids, about 45 to about 100 amino acids, about 45 to about 90 amino acids, about 45 to about 80 amino acids, about 45 to about 70 amino acids, about 45 to about 60 amino acids, about 45 to about 50 amino acids, about 50 to about 100 amino acids, It may have about 50 to about 90 amino acids, about 50 to about 80 amino acids, about 50 to about 70 amino acids, about 50 to about 60 amino acids, about 60 to about 100 amino acids, about 60 to about 90 amino acids, about 60 to about 80 amino acids, about 60 to about 70 amino acids, about 70 to about 100 amino acids, about 70 to about 90 amino acids, about 70 to about 80 amino acids, about 80 to about 100 amino acids, about 80 to about 90 amino acids, or about 90 to about 100 amino acids.

[0060] In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGSG sequences.

[0061] In some embodiments, the linker sequence may comprise or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 7). In some embodiments, the linker sequence may be encoded by a nucleic acid comprising or consisting of GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 8). In some embodiments, the linker sequence may comprise or consist of GGGSGGGS (SEQ ID NO: 9).

[0062] Target Binding Domain In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the additional one or more target binding domains are selected from the group consisting of an antigen binding domain (e.g., any of the exemplary antigen binding domains described herein or known in the art), a soluble interleukin, or It can be a cytokine protein (e.g., any of the exemplary soluble interleukin proteins or soluble cytokine proteins described herein), and a soluble interleukin or cytokine receptor (e.g., any of the exemplary soluble interleukin receptors or soluble cytokine receptors described herein).

[0063] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary first target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary second target binding domains described herein or known in the art), and the one or more additional target binding domains are each independently selected from the group consisting of CD16a, CD28, CD3 (e.g., CD3α, CD3β, CD3δ, CD3M, and CD3β). 3Κ), CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TN Fα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACA M5, UL16-binding proteins (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor , IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

[0064] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the one or more additional target binding domains each independently have a total amino acid sequence of from about 5 to about 1000 amino acids, from about 5 to about 950 amino acids, from about 5 to about 900 amino acids, from about 5 to about 850 amino acids, from about 5 to about 800 amino acids, from about 5 to about 750 amino acids, from about 5 to about 700 amino acids, from about 5 to about 650 amino acids, from about 5 to about 600 amino acids, from about 5 to about 550 amino acids, from about 5 to about 500 amino acids, from about 5 to about 450 amino acids, from about 5 to about 400 amino acids, from about 5 to about 350 amino acids, from about 5 to about 300 amino acids, from about 5 to about 280 amino acids, from about 5 to about 260 amino acids, from about 5 to about 5 amino acids, about 5 to about 240 amino acids, about 5 to about 220 amino acids, about 5 to about 200 amino acids, about 5 to about 195 amino acids, about 5 to about 190 amino acids, about 5 to about 185 amino acids, about 5 to about 180 amino acids, about 5 to about 175 amino acids, about 5 to about 170 amino acids, about 5 to about 165 amino acids, about 5 to about 160 amino acids, about 5 to about 155 amino acids, about 5 to about 150 amino acids, about 5 to about 145 amino acids, about 5 to about 140 amino acids, about 5 to about 135 amino acids, about 5 to about 130 amino acids, about 5 to about 125 amino acids, about 5 to about 120 amino acids, about 5 to about 115 amino acids, about 5 to about 110 amino acids, about 5 to about 105 amino acids, about 5 to about 100 amino acids, about 5 to about 95 amino acids, Amino acids up to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids, about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino acids, 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to about 80 amino acids,About 10 to about 75 amino acids, about 10 to about 70 amino acids, about 10 to about 65 amino acids, about 10 to about 60 amino acids, about 10 to about 55 amino acids, about 10 to about 50 amino acids, about 10 to about 45 amino acids, about 10 to about 40 amino acids, about 10 to about 35 amino acids, about 10 to about 30 amino acids, about 10 to about 25 amino acids, about 10 to about 20 amino acids, about 10 to about 15 amino acids, about 15 to about 1000 amino acids, about 15 to about 950 amino acids, about 15 amino acids to about 900 amino acids, about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700 amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to about 600 amino acids, about 15 amino acids to about 550 amino acids, about 15 amino acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15 amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids, about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino acids, about 15 amino acids to about 260 amino acids, about 1 5 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids amino acids, about 15 to about 135 amino acids, about 15 to about 130 amino acids, about 15 to about 125 amino acids, about 15 to about 120 amino acids, about 15 to about 115 amino acids, about 15 to about 110 amino acids, about 15 to about 105 amino acids, about 15 to about 100 amino acids, about 15 to about 95 amino acids, about 15 to about 90 amino acids, about 15 to about 85 amino acids, about 15 to about 80 amino acids, about 15 to about 75 amino acids, about 15 to about 70 amino acids, about 15 to about 65 amino acids,about, 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 1000 amino acids, about 20 amino acids to about 950 amino acids, about 20 amino acids to about 900 amino acids, about 20 amino acids to about 850 amino acids, about 20 amino acids to about 800 amino acids, about 20 amino acids to about 750 amino acids, about 20 amino acids to about 1000 amino acids amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids , about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 12 0 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids,About 20 to about 40 amino acids, about 20 to about 35 amino acids, about 20 to about 30 amino acids, about 20 to about 25 amino acids, about 25 to about 1000 amino acids, about 25 to about 950 amino acids, about 25 to about 900 amino acids, about 25 to about 850 amino acids, about 25 to about 800 amino acids, about 25 to about 750 amino acids, about 25 to about 700 amino acids, about 25 to about 650 amino acids, about 25 to about 600 amino acids, about 25 to about 550 amino acids, about 25 to about 500 amino acids amino acids, about 25 to about 450 amino acids, about 25 to about 400 amino acids, about 25 to about 350 amino acids, about 25 to about 300 amino acids, about 25 to about 280 amino acids, about 25 to about 260 amino acids, about 25 to about 240 amino acids, about 25 to about 220 amino acids, about 25 to about 200 amino acids, about 25 to about 195 amino acids, about 25 to about 190 amino acids, about 25 to about 185 amino acids, about 25 to about 180 amino acids, about 25 to about 175 amino acids, about 25 to about 1 70 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 25 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids from about 95 amino acids, from about 25 amino acids to about 90 amino acids, from about 25 amino acids to about 85 amino acids, from about 25 amino acids to about 80 amino acids, from about 25 amino acids to about 75 amino acids, from about 25 amino acids to about 70 amino acids, from about 25 amino acids to about 65 amino acids, from about 25 amino acids to about 60 amino acids, from about 25 amino acids to about 55 amino acids, from about 25 amino acids to about 50 amino acids, from about 25 amino acids to about 45 amino acids, from about 25 amino acids to about 40 amino acids, from about 25 amino acids to about 35 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 1000 amino acids, from about 30 amino acids to about 950 amino acids, amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids, about 30 amino acids to about 300 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, About 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 to about 105 amino acids, about 30 to about 100 amino acids, about 30 to about 95 amino acids, about 30 to about 90 amino acids, about 30 to about 85 amino acids, about 30 to about 80 amino acids, about 30 to about 75 amino acids, about 30 to about 70 amino acids, about 30 to about 65 amino acids, about 30 to about 60 amino acids, about 30 to about 55 amino acids, about 30 to about 50 amino acids, about 30 to about 45 amino acids, about 30 to about 40 amino acids, about 30 to about 35 amino acids amino acids, about 35 to about 1000 amino acids, about 35 to about 950 amino acids, about 35 to about 900 amino acids, about 35 to about 850 amino acids, about 35 to about 800 amino acids, about 35 to about 750 amino acids, about 35 to about 700 amino acids, about 35 to about 650 amino acids, about 35 to about 600 amino acids, about 35 to about 550 amino acids, about 35 to about 500 amino acids, about 35 to about 450 amino acids, about 35 to about 400 amino acids, about 35 to about 350 amino acids, about 35 amino acids about 300 amino acids, about 35 amino acids to about 280 amino acids, about 35 amino acids to about 260 amino acids, about 35 amino acids to about 240 amino acids, about 35 amino acids to about 220 amino acids, about 35 amino acids to about 200 amino acids, about 35 amino acids to about 195 amino acids, about 35 amino acids to about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino acids to about 180 amino acids, about 35 amino acids to about 175 amino acids, about 35 amino acids to about 170 amino acids, about 35 amino acids to about 165 amino acids, about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino acids,about 35 amino acids to about 150 amino acids, about 35 amino acids to about 145 amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 125 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 ... amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 1000 amino acids, about 40 amino acids to about 950 amino acids, about 40 amino acids to about 900 amino acids, about 40 amino acids to about 850 amino acids, about 40 amino acids to about 800 amino acids, about 40 amino acids to about 750 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about About 350 amino acids, about 40 to about 300 amino acids, about 40 to about 280 amino acids, about 40 to about 260 amino acids, about 40 to about 240 amino acids, about 40 to about 220 amino acids, about 40 to about 200 amino acids, about 40 to about 195 amino acids, about 40 to about 190 amino acids, about 40 to about 185 amino acids, about 40 to about 180 amino acids, about 40 to about 175 amino acids, about 40 to about 170 amino acids, about 40 to about 165 amino acids, about 40 to about 160 amino acids , about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to about 1000 amino acids about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45 amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids, about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino acids, about 45 amino acids to about 300 amino acids, about 45 amino acids to about 280 amino acids, about 45 amino acids to about 260 amino acids, about 45 amino acids to about 240 amino acids, about 45 amino acids to about 220 amino acids, about 45 amino acids to about 200 amino acids, about 45 amino acids to about 195 amino acids,About 45 amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids, about 45 amino acids to about 1 20 amino acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 75 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 65 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 55 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 1000 amino acids amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 3 ... amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids,Approximately 50 amino acids to approximately 145 amino acids, approximately 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, 0 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids, about 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids amino acids, about 55 amino acids to about 400 amino acids, about 55 amino acids to about 350 amino acids, about 55 amino acids to about 300 amino acids, about 55 amino acids to about 280 amino acids, about 55 amino acids to about 260 amino acids, about 55 amino acids to about 240 amino acids, about 55 amino acids to about 220 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 amino acids to about 17 ... amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids,About 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 60 0 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60 amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, About 60 to about 175 amino acids, about 60 to about 170 amino acids, about 60 to about 165 amino acids, about 60 to about 160 amino acids, about 60 to about 155 amino acids, about 60 to about 150 amino acids, about 60 to about 145 amino acids, about 60 to about 140 amino acids, about 60 to about 135 amino acids, about 60 to about 130 amino acids, about 60 to about 125 amino acids, about 60 to about 120 amino acids, about 60 to about 115 amino acids amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 65 amino acids, about 65 amino acids to about 1000 amino acids, about 65 amino acids to about 950 amino acids, about 65 amino acids to about 900 amino acids, About 65 amino acids to about 850 amino acids, about 65 amino acids to about 800 amino acids, about 65 amino acids to about 750 amino acids, about 65 amino acids to about 700 amino acids, about 65 amino acids to about 650 amino acids, about 65 amino acids to about 600 amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to about 500 amino acids, about 65 amino acids to about 450 amino acids, about 65 amino acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65 amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids, about 65 amino acids to about 260 amino acids, about 65 amino acids to about 24 0 amino acids, about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 14 ... amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 1000 amino acids from about 70 amino acids to about 950 amino acids, from about 70 amino acids to about 900 amino acids, from about 70 amino acids to about 850 amino acids, from about 70 amino acids to about 800 amino acids, from about 70 amino acids to about 750 amino acids, from about 70 amino acids to about 700 amino acids, from about 70 amino acids to about 650 amino acids, from about 70 amino acids to about 600 amino acids, from about 70 amino acids to about 550 amino acids, from about 70 amino acids to about 500 amino acids, from about 70 amino acids to about 450 amino acids, from about 70 amino acids to about 400 amino acids, from about 70 amino acids to about 350 amino acids, from about 70 amino acids to about 300 amino acids, from about 70 amino acids to about 280 amino acids,About 70 amino acids to about 260 amino acids, about 70 amino acids to about 240 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 14 5 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 10 00 amino acids, about 75 amino acids to about 950 amino acids, about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino acids, about 75 amino acids to about 800 amino acids, about 75 amino acids to about 750 amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75 amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids to about 350 amino acids, about 75 amino acids to about 3 ... about 75 to about 280 amino acids, about 75 to about 260 amino acids, about 75 to about 240 amino acids, about 75 to about 220 amino acids, about 75 to about 200 amino acids, about 75 to about 195 amino acids, about 75 to about 190 amino acids, about 75 to about 185 amino acids, about 75 to about 180 amino acids, about 75 to about 175 amino acids, about 75 to about 170 amino acids, about 75 to about 165 amino acids, about 75 to about 160 amino acids, about 75 to about 155 amino acids, about 75 to about 150 amino acids,Approximately 75 to 145 amino acids, About 75 amino acids to about 140 amino acids, about 75 amino acids to about 135 amino acids, about 75 amino acids to about 130 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 1000 amino acids, about 80 amino acids to about 950 amino acids amino acids, about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino acids, about 80 amino acids to about 800 amino acids, about 80 amino acids to about 750 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids, about 80 amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids, about 80 amino acids to about 350 amino acids, about 80 amino acids to about 300 amino acids, about 80 amino acids to about 280 amino acids, about 80 amino acids to about 260 amino acids, about 80 amino acids to about 240 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 Amino acids up to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 85 amino acids to about 1000 amino acids, about 85 amino acids to about 950 amino acids, about 85 amino acids to about 900 amino acids,About 85 amino acids to about 850 amino acids, about 85 amino acids to about 800 amino acids, about 85 amino acids to about 750 amino acids, about 85 amino acids to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 26 ... About 240 amino acids, about 85 to about 220 amino acids, about 85 to about 200 amino acids, about 85 to about 195 amino acids, about 85 to about 190 amino acids, about 85 to about 185 amino acids, about 85 to about 180 amino acids, about 85 to about 175 amino acids, about 85 to about 170 amino acids, about 85 to about 165 amino acids, about 85 to about 160 amino acids, about 85 to about 155 amino acids, about 85 to about 150 amino acids, about 85 to about 145 amino acids, about 85 to about 140 amino acids , about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85 amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids, about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90 amino acids to about 850 amino acids, about 90 amino acids to about 1000 amino acids About 800 amino acids, about 90 to about 750 amino acids, about 90 to about 700 amino acids, about 90 to about 650 amino acids, about 90 to about 600 amino acids, about 90 to about 550 amino acids, about 90 to about 500 amino acids, about 90 to about 450 amino acids, about 90 to about 400 amino acids, about 90 to about 350 amino acids, about 90 to about 300 amino acids, about 90 to about 280 amino acids, about 90 to about 260 amino acids, about 90 to about 240 amino acids, about 90 to about 220 amino acids,Approximately 90 to 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 1000 amino acids, about 95 amino acids to about 950 amino acids, about 95 amino acids to about 900 amino acids, about 95 amino acids to about 850 amino acids, about 95 amino acids to about 800 amino acids, about 95 amino acids to about 750 amino acids, about 95 amino acids to about 700 amino acids, about 95 amino acids to about 650 amino acids, about 95 amino acids to about 600 amino acids, about 95 amino acids to about 550 amino acids, about 95 amino acids to about 500 amino acids, about 95 amino acids to about 450 amino acids, about 95 amino acids to about 400 amino acids, about 95 amino acids to about 350 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 280 amino acids, about 95 amino acids to about 260 amino acids, about 95 amino acids to about 240 amino acids, about 95 amino acids to about 220 amino acids, About 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids to about 135 amino acids, about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 to about 600 amino acids, about 100 to about 550 amino acids, about 100 to about 500 amino acids, about 100 to about 450 amino acids, about 100 to about 400 amino acids, about 100 to about 350 amino acids, about 100 to about 300 amino acids, about 100 to about 280 amino acids, about 100 to about 260 amino acids, about 100 to about 240 amino acids, about 100 to about 220 amino acids, about 100 to about 200 amino acids, about 100 to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids,about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 1000 amino acids, about 105 amino acids to about 950 amino acids, about 105 amino acids to about 900 amino acids, about 105 amino acids to about 850 amino acids, about 105 amino acids to about 800 amino acids, about 105 amino acids to about 750 amino acids, about 105 amino acids to about 700 amino acids, about 105 amino acids to about 650 amino acids, about 105 amino acids to about 600 amino acids, about 105 amino acids to about 550 amino acids, about 105 amino acids to about 500 amino acids, about 105 amino acids to about 450 amino acids, About 105 amino acids to about 400 amino acids, about 105 amino acids to about 350 amino acids, about 105 amino acids to about 300 amino acids, about 105 amino acids to about 280 amino acids, about 105 amino acids to about 260 amino acids, about 105 amino acids to about 240 amino acids, about 105 amino acids to about 220 amino acids, about 105 amino acids to about 200 amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 Amino acids, about 105 to about 165 amino acids, about 105 to about 160 amino acids, about 105 to about 155 amino acids, about 105 to about 150 amino acids, about 105 to about 145 amino acids, about 105 to about 140 amino acids, about 105 to about 135 amino acids, about 105 to about 130 amino acids, about 105 to about 125 amino acids, about 105 to about 120 amino acids, about 105 to about 115 amino acids, about 105 to about 110 amino acids, about 110 to about 1000 amino acids, about 110 amino acids about 950 amino acids, about 110 amino acids to about 900 amino acids, about 110 amino acids to about 850 amino acids, about 110 amino acids to about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110 amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids, about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550 amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to about 450 amino acids, about 110 amino acids to about 400 amino acids, about 110 amino acids to about 350 amino acids, about 110 amino acids to about 300 amino acids, about 110 amino acids to about 280 amino acids, about 110 amino acids to about 260 amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids,About 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115 amino acids to about 800 amino acids, about 115 amino acids to about 750 Amino acids, about 115 amino acids to about 700 amino acids, about 115 amino acids to about 650 amino acids, about 115 amino acids to about 600 amino acids, about 115 amino acids to about 550 amino acids, about 115 amino acids to about 500 amino acids, about 115 amino acids to about 450 amino acids, about 115 amino acids to about 400 amino acids, about 115 amino acids to about 350 amino acids, about 115 amino acids to about 300 amino acids, about 115 amino acids to about 280 amino acids, about 115 amino acids to about 260 amino acids, about 115 amino acids to about 240 amino acids, about 115 amino acids to about 2 ... about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids, about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to about 600 amino acids, about 120 amino acids to about 550 amino acids, about 120 amino acids to about 500 amino acids,About 120 amino acids to about 450 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350 amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids to about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120 amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids Amino acids, about 120 to about 170 amino acids, about 120 to about 165 amino acids, about 120 to about 160 amino acids, about 120 to about 155 amino acids, about 120 to about 150 amino acids, about 120 to about 145 amino acids, about 120 to about 140 amino acids, about 120 to about 135 amino acids, about 120 to about 130 amino acids, about 120 to about 125 amino acids, about 125 to about 1000 amino acids, about 125 to about 950 amino acids, about 125 to about 900 amino acids, about 125 amino acids about 125 to about 850 amino acids, about 125 to about 800 amino acids, about 125 to about 750 amino acids, about 125 to about 700 amino acids, about 125 to about 650 amino acids, about 125 to about 600 amino acids, about 125 to about 550 amino acids, about 125 to about 500 amino acids, about 125 to about 450 amino acids, about 125 to about 400 amino acids, about 125 to about 350 amino acids, about 125 to about 300 amino acids, about 125 to about 280 amino acids, about 125 to about 260 amino acids, about 125 Amino acids up to about 240 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids,About 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 1000 amino acids, about 130 amino acids to about 950 amino acids, about 130 amino acids to about 900 amino acids, about 130 amino acids to about 850 amino acids, about 130 amino acids to about 800 amino acids, about 130 amino acids to about 750 amino acids, about 130 amino acids to about 700 amino acids, about 130 amino acids to about 650 amino acids, about 130 amino acids to about 600 amino acids, about 130 amino acids to about 550 amino acids, about 130 amino acids to about 500 Amino acids, about 130 amino acids to about 450 amino acids, about 130 amino acids to about 400 amino acids, about 130 amino acids to about 350 amino acids, about 130 amino acids to about 300 amino acids, about 130 amino acids to about 280 amino acids, about 130 amino acids to about 260 amino acids, about 130 amino acids to about 240 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 18 ...80 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 1 About 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids, about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900 amino acids, about 135 amino acids to about 850 amino acids, about 135 amino acids to about 800 amino acids, about 135 Amino acids up to about 750 amino acids, about 135 amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids, about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550 amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to about 450 amino acids, about 135 amino acids to about 400 amino acids, about 135 amino acids to about 350 amino acids, about 135 amino acids to about 300 amino acids, about 135 amino acids to about 280 amino acids, about 135 amino acids to about 260 amino acids, about 135 amino acids to about 240 amino acids, about 135 amino acids to about 220 amino acids,About 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 1000 Amino acids, about 140 amino acids to about 950 amino acids, about 140 amino acids to about 900 amino acids, about 140 amino acids to about 850 amino acids, about 140 amino acids to about 800 amino acids, about 140 amino acids to about 750 amino acids, about 140 amino acids to about 700 amino acids, about 140 amino acids to about 650 amino acids, about 140 amino acids to about 600 amino acids, about 140 amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 350 amino acids, about 140 amino acids to about About 300 amino acids, about 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids, about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900 amino acids, about 145 amino acids to about 850 amino acids, about 145 amino acids to about 800 amino acids, about 145 amino acids to about 750 amino acids, about 145 amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids, about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550 amino acids, about 145 amino acids to about 500 amino acids,About 145 amino acids to about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145 amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids, about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260 amino acids, about 145 amino acids to about 240 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 200 amino acids, About 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 Amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 26 ...60 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 2 About 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 1000 amino acids, about 155 amino acids to about 950 amino acids, about 155 Amino acids up to about 900 amino acids, about 155 amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids, about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700 amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155 amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids, about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350 amino acids, about 155 amino acids to about 300 amino acids, about 155 amino acids to about 280 amino acids,About 155 amino acids to about 260 amino acids, about 155 amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to about 950 amino acids Amino acids, about 160 amino acids to about 900 amino acids, about 160 amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids, about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700 amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids, about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350 amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to about About 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 1000 amino acids, about 165 amino acids to about 950 amino acids, about 165 Amino acids up to about 900 amino acids, about 165 amino acids to about 850 amino acids, about 165 amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids, about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650 amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165 amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids, about 165 amino acids to about 350 amino acids, about 165 amino acids to about 300 amino acids, about 165 amino acids to about 280 amino acids,About 165 amino acids to about 260 amino acids, about 165 amino acids to about 240 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 1000 amino acids, about 170 amino acids to about 950 amino acids, about 170 amino acids to about 900 amino acids, about 170 amino acids to about 850 Amino acids, about 170 amino acids to about 800 amino acids, about 170 amino acids to about 750 amino acids, about 170 amino acids to about 700 amino acids, about 170 amino acids to about 650 amino acids, about 170 amino acids to about 600 amino acids, about 170 amino acids to about 550 amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170 amino acids to about 350 amino acids, about 170 amino acids to about 300 amino acids, about 170 amino acids to about 280 amino acids, about 170 amino acids to about 26 ... About 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175 amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids, about 175 amino acids to about 800 amino acids, about 175 amino acids to about 750 amino acids, about 175 Amino acids to about 700 amino acids, about 175 amino acids to about 650 amino acids, about 175 amino acids to about 600 amino acids, about 175 amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids, about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175 amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 200 amino acids,About 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids, about 180 amino acids to about 950 amino acids, about 180 amino acids to about 900 amino acids, about 180 amino acids to about 850 amino acids, about 180 amino acids to about 800 amino acids, about 180 amino acids to about 750 amino acids, about 180 amino acids to about 700 amino acids, about 180 amino acids to about 650 amino acids , about 180 amino acids to about 600 amino acids, about 180 amino acids to about 550 amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to about 450 amino acids, about 180 amino acids to about 400 amino acids, about 180 amino acids to about 350 amino acids, about 180 amino acids to about 300 amino acids, about 180 amino acids to about 280 amino acids, about 180 amino acids to about 260 amino acids, about 180 amino acids to about 240 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 200 amino acids, About 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 1000 amino acids, about 185 amino acids to about 950 amino acids, about 185 amino acids to about 900 amino acids, about 185 amino acids to about 850 amino acids, about 185 amino acids to about 800 amino acids, about 185 amino acids to about 750 amino acids, about 185 amino acids to about 700 amino acids, about 185 amino acids to about 650 amino acids, about 185 amino acids to about 600 amino acids , about 185 amino acids to about 550 amino acids, about 185 amino acids to about 500 amino acids, about 185 amino acids to about 450 amino acids, about 185 amino acids to about 400 amino acids, about 185 amino acids to about 350 amino acids, about 185 amino acids to about 300 amino acids, about 185 amino acids to about 280 amino acids, about 185 amino acids to about 260 amino acids, about 185 amino acids to about 240 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, About 185 amino acids to about 190 amino acids, about 190 amino acids to about 1000 amino acids, about 190 amino acids to about 950 amino acids, about 190 amino acids to about 900 amino acids, about 190 amino acids to about 850 amino acids, about 190 amino acids to about 800 amino acids, about 190 amino acids to about 750 amino acids, about 190 amino acids to about 700 amino acids, about 190 amino acids to about 650 amino acids, about 190 amino acids to about 600 amino acids, about 190 amino acids to about 550 amino acids, about 190 amino acids to about 500 amino acids, about 190 amino acids to about 450 amino acids, about 190 amino acids to about 400 Amino acids, about 190 amino acids to about 350 amino acids, about 190 amino acids to about 300 amino acids, about 190 amino acids to about 280 amino acids, about 190 amino acids to about 260 amino acids, about 190 amino acids to about 240 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 1000 amino acids, about 195 amino acids to about 950 amino acids, about 195 amino acids to about 900 amino acids, about 195 amino acids to about 850 amino acids, about 195 amino acids to about 800 amino acids, about 195 amino acids to about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to about 240 amino acids, about 195 amino acids to about 220 amino acids, about 195 Amino acids to about 200 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids,About 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 Amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 450 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 350 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 Amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 450 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 350 amino acids,About 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 450 Amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 350 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about About 500 amino acids, about 300 to about 450 amino acids, about 300 to about 400 amino acids, about 300 to about 350 amino acids, about 350 to about 1000 amino acids, about 350 to about 950 amino acids, about 350 to about 900 amino acids, about 350 to about 850 amino acids, about 350 to about 800 amino acids, about 350 to about 750 amino acids, about 350 to about 700 amino acids, about 350 to about 650 amino acids, about 350 to about 600 amino acids, about 350 to about 550 amino acids, about 350 Amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids,About 400 amino acids to about 450 amino acids, about 450 amino acids to about 1000 amino acids, about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 Amino acids, about 500 to about 900 amino acids, about 500 to about 850 amino acids, about 500 to about 800 amino acids, about 500 to about 750 amino acids, about 500 to about 700 amino acids, about 500 to about 650 amino acids, about 500 to about 600 amino acids, about 500 to about 550 amino acids, about 550 to about 1000 amino acids, about 550 to about 950 amino acids, about 550 to about 900 amino acids, about 550 to about 850 amino acids, about 550 to about 800 amino acids, about 550 to about About 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 Amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids,It may have about 750 to about 850 amino acids, about 750 to about 800 amino acids, about 800 to about 1000 amino acids, about 800 to about 950 amino acids, about 800 to about 900 amino acids, about 800 to about 850 amino acids, about 850 to about 1000 amino acids, about 850 to about 950 amino acids, about 850 to about 900 amino acids, about 900 to about 1000 amino acids, about 900 to about 950 amino acids, or about 950 to about 1000 amino acids.

[0065] 1×10 of any of the target binding domains described herein -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M or 1 x 10 -13 The dissociation equilibrium constant (K D In some embodiments, the antigen binding protein constructs provided herein can bind to their targets at 1 x 10 -3 M ~ approx. 1×10 -5 M, about 1 x 10 -4 M ~ approx. 1×10 -6 M, about 1 x 10 -5 M ~ approx. 1×10 -7 M, about 1 x 10 -6 M ~ approx. 1×10 -8 M, about 1 x 10 -7 M ~ approx. 1×10 -9 M, about 1 x 10 -8 M ~ approx. 1×10 -10 M, or approximately 1 x 10 -9 M ~ approx. 1×10 -11 K of M (including its upper and lower bounds) D It can bind to a specific antigen.

[0066] Any of the target binding domains described herein may be administered at a concentration of from about 1 pM to about 30 nM (e.g., from about 1 pM to about 25 nM, from about 1 pM to about 20 nM, from about 1 pM to about 15 nM, from about 1 pM to about 10 nM, from about 1 pM to about 5 nM, from about 1 pM to about 2 nM, from about 1 pM to about 1 nM, from about 1 pM to about 950 pM, from about 1 pM to about 900 pM, 1pM to about 850pM, about 1pM to about 800pM, about 1pM to about 750pM, about 1pM to about 700pM, about 1pM to about 650pM, about 1pM to about 600pM , about 1 pM to about 550 pM, about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM, approximately 1 pM to approximately 250 pM, approximately 1 pM to approximately 200 pM, approximately 1 pM to approximately 150 pM, approximately 1 pM to approximately 100 pM, approximately 1 pM to approximately 90 pM, approximately 1 pM to approximately 80 pM, approximately 1 pM to approximately 70 pM, approximately 1 pM to approximately 60 pM, approximately 1 pM to approximately 50 pM, approximately 1 pM to approximately 40 pM, approximately 1 pM to approximately 3 0pM, approximately 1pM to approximately 20pM, approximately 1pM to approximately 10pM, approximately 1pM to approximately 5pM, approximately 1pM to approximately 4pM, approximately 1pM to approximately 3pM, approximately 1pM to approximately 2pM, approximately 2pM to approximately 30nM, approximately 2pM to approximately 25nM, approximately 2pM to approximately 20nM, approximately 2pM to approximately 15nM, approximately 2pM to approximately 10nM, approximately 2pM ~Approximately 5 nm, approximately 2 pM~approximately 2 nm, approximately 2 pM~approximately 1 nm, approximately 2 pM~approximately 950 pM, approximately 2 pM~approximately 900 pM, approximately 2 pM~approximately 850 pM, approximately 2 pM~approximately 800 pM, approximately 2 pM~approximately 750 pM, approximately 2 pM~approximately 700 pM, approximately 2 pM~approximately 650 pM, approximately 2 pM~approximately 600 pM, approximately 2 p M~approx. 550pM, approx. 2pM~approx. 500pM, approx. 2pM~approx. 450pM, approx. 2pM~approx. 400pM, approx. 2pM~approx. 350pM, approx. 2pM~approx. 300pM, approx. 2pM~approx. 250pM, approx. 2pM~approx. 200pM, approx. 2pM~approx. 150pM, approx. 2pM~approx. 100pM, approx. 2pM~approx. 900pM pM, approximately 2pM to approximately 80pM, approximately 2pM to approximately 70pM, approximately 2pM to approximately 60pM, approximately 2pM to approximately 50pM, approximately 2pM to approximately 40pM, approximately 2pM to approximately 30pM, approximately 2pM to approximately 20pM, approximately 2pM to approximately 10pM, approximately 2pM to approximately 5pM, approximately 2pM to approximately 4pM, approximately 2pM to approximately 3pM, approximately 5pM M ~ approximately 30 nmM, approximately 5 pM ~ approximately 25 nmM, approximately 5 pM ~ approximately 20 nmM, approximately 5 pM ~ approximately 15 nmM, approximately 5 pM ~ approximately 10 nmM, approximately 5 pM ~ approximately 5 nmM, approximately 5 pM ~ approximately 2 nmM, approximately 5 pM ~ approximately 1 nmM, approximately 5 pM ~ approximately 950 nmM, approximately 5 pM ~ approximately 900 nmM, approximately 5 pM ~ approximately 850 nmM, approximately 5 pM ~ approximately 8 0pM, approximately 5pM to approximately 750pM, approximately 5pM to approximately 700pM, approximately 5pM to approximately 650pM, approximately 5pM to approximately 600pM, approximately 5pM to approximately 550pM, approximately 5pM to approximately 500pM, approximately 5pM to approximately 450pM, approximately 5pM to approximately 400pM, approximately 5pM to approximately 350pM, approximately 5pM to approximately 300pM Approximately 5 pM to approximately 250 pM, approximately 5 pM to approximately 200 pM, approximately 5 pM to approximately 150 pM, approximately 5 pM to approximately 100 pM, approximately 5 pM to approximately 90 pM, approximately 5 pM to approximately 80 pM, approximately 5 pM to approximately 70 pM, approximately 5 pM to approximately 60 pM, approximately 5 pM to approximately 50 pM, approximately 5 pM to approximately 40 pM, approximately 5 pM to approximately 30 pM.Approximately 5 pM to approximately 20 pM, approximately 5 pM to approximately 10 pM, approximately 10 pM to approximately 30 nm, approximately 10 pM to approximately 25 nm, approximately 10 pM to approximately 20 nm, approximately 10 pM to approximately 15 nm, approximately 10 pM to approximately 10 nm, approximately 10 pM to approximately 5 nm, approximately 10 pM to approximately 2 nm, approximately 10 pM to approximately 1 nm, approximately 10 pM to approximately 950 nm pM, approximately 10 pM to approximately 900 pM, approximately 10 pM to approximately 850 pM, approximately 10 pM to approximately 800 pM, approximately 10 pM to approximately 750 pM, approximately 10 pM to approximately 700 pM, approximately 10 pM to approximately 650 pM, approximately 10 pM to approximately 600 pM, approximately 10 pM to approximately 550 pM, approximately 10 pM to approximately 500 pM, approximately 10 pM ~approximately 450pM, approximately 10pM~approximately 400pM, approximately 10pM~approximately 350pM, approximately 10pM~approximately 300pM, approximately 10pM~approximately 250pM, approximately 10pM~approximately 200pM, approximately 10pM~approximately 150pM, approximately 10pM~approximately 100pM, approximately 10pM~approximately 90pM, approximately 10pM~approximately 80pM, approximately 1 0pM to approximately 70pM, approximately 10pM to approximately 60pM, approximately 10pM to approximately 50pM, approximately 10pM to approximately 40pM, approximately 10pM to approximately 30pM, approximately 10pM to approximately 20pM, approximately 15pM to approximately 30nM, approximately 15pM to approximately 25nM, approximately 15pM to approximately 20nM, approximately 15pM to approximately 15nM, approximately 15pM to approximately 10 nm, approximately 15 pM to approximately 5 nm, approximately 15 pM to approximately 2 nm, approximately 15 pM to approximately 1 nm, approximately 15 pM to approximately 950 pM, approximately 15 pM to approximately 900 pM, approximately 15 pM to approximately 850 pM, approximately 15 pM to approximately 800 pM, approximately 15 pM to approximately 750 pM, approximately 15 pM to approximately 700 pM, approximately 15 pM to approximately 65 0pM, approximately 15pM to approximately 600pM, approximately 15pM to approximately 550pM, approximately 15pM to approximately 500pM, approximately 15pM to approximately 450pM, approximately 15pM to approximately 400pM, approximately 15pM to approximately 350pM, approximately 15pM to approximately 300pM, approximately 15pM to approximately 250pM, approximately 15pM to approximately 200pM, approximately 15pM M~approx. 150pM, 15pM~approx. 100pM, 15pM~approx. 90pM, 15pM~approx. 80pM, 15pM~approx. 70pM, 15pM~approx. 60pM, 15pM~approx. 50pM, 15pM~approx. 40pM, 15pM~approx. 30pM, 15pM~approx. 20pM, 20pM~approx. 30 nm, approximately 20 pM to approximately 25 nm, approximately 20 pM to approximately 20 nm, approximately 20 pM to approximately 15 nm, approximately 20 pM to approximately 10 nm, approximately 20 pM to approximately 5 nm, approximately 20 pM to approximately 2 nm, approximately 20 pM to approximately 1 nm, approximately 20 pM to approximately 950 nm, approximately 20 pM to approximately 900 nm, approximately 20 pM to approximately 850 nm.Approximately 2 0pM~approx. 800pM, approx. 20pM~approx. 750pM, approx. 20pM~approx. 700pM, approx. 20pM~approx. 650pM, approx. 20pM~approx. 600pM, approx. 20pM~approx. 550pM, approx. 20pM~approx. 500pM, approx. 20pM~approx. 450pM, approx. 20pM~approx. 400pM, approx. 20pM~approx. 3 50pM, approximately 20pM to approximately 300pM, approximately 20pM to approximately 250pM, approximately 20pM to approximately 20pM, approximately 200pM to approximately 150pM, approximately 20pM to approximately 100pM, approximately 20pM to approximately 90pM, approximately 20pM to approximately 80pM, approximately 20pM to approximately 70pM, approximately 20pM to approximately 60pM, approximately 20pM~ Approximately 50 pM, approximately 20 pM to approximately 40 pM, approximately 20 pM to approximately 30 pM, approximately 30 pM to approximately 30 nm, approximately 30 pM to approximately 25 nm, approximately 30 pM to approximately 30 nm, approximately 30 pM to approximately 15 nm, approximately 30 pM to approximately 10 nm, approximately 30 pM to approximately 5 nm, approximately 30 pM to approximately 2 nm, approximately 30 pM to approximately 1 nm, approximately 30pM to approximately 950pM, approximately 30pM to approximately 900pM, approximately 30pM to approximately 850pM, approximately 30pM to approximately 800pM, approximately 30pM to approximately 750pM, approximately 30pM to approximately 700pM, approximately 30pM to approximately 650pM, approximately 30pM to approximately 600pM, approximately 30pM to approximately 550pM, approximately 30pM to approximately 5 00pM, approximately 30pM to approximately 450pM, approximately 30pM to approximately 400pM, approximately 30pM to approximately 350pM, approximately 30pM to approximately 300pM, approximately 30pM to approximately 250pM, approximately 30pM to approximately 200pM, approximately 30pM to approximately 150pM, approximately 30pM to approximately 100pM, approximately 30pM to approximately 90pM, approximately 3 0pM~approx. 80pM, approx. 30pM~approx. 70pM, approx. 30pM~approx. 60pM, approx. 30pM~approx. 50pM, approx. 30pM~approx. 40pM, approx. 40pM~approx. 30nM, approx. 40pM~approx. 15nM, approx. 40pM~approx. 10nM, approx. 40pM~ Approximately 5 nm, approximately 40 pM to approximately 2 nm, approximately 40 pM to approximately 1 nm, approximately 40 pM to approximately 950 pM, approximately 40 pM to approximately 900 pM, approximately 40 pM to approximately 850 pM, approximately 40 pM to approximately 800 pM, approximately 40 pM to approximately 750 pM, approximately 40 pM to approximately 700 pM, approximately 40 pM to approximately 650 pM, approximately 40 pM~ Approximately 600pM, approximately 40pM to approximately 550pM, approximately 40pM to approximately 500pM, approximately 40pM to approximately 450pM, approximately 40pM to approximately 400pM, approximately 40pM to approximately 350pM, approximately 40pM to approximately 300pM, approximately 40pM to approximately 250pM, approximately 40pM to approximately 200pM, approximately 40pM to approximately 150pMApproximately 40 pM to approximately 100 pM, approximately 40 pM to approximately 90 pM, approximately 40 pM to approximately 80 pM, approximately 40 pM to approximately 70 pM, approximately 40 pM to approximately 60 pM, approximately 40 pM to approximately 50 pM, approximately 50 pM to approximately 30 nM, approximately 50 pM to approximately 25 nM, approximately 50 pM to approximately 30 nM, approximately 50 pM to approximately 15 nM, approximately 50 pM ~ approximately 10 nmM, approximately 50 pM ~ approximately 5 nmM, approximately 50 pM ~ approximately 2 nmM, approximately 50 pM ~ approximately 1 nmM, approximately 50 pM ~ approximately 950 pM, approximately 50 pM ~ approximately 900 pM, approximately 50 pM ~ approximately 850 pM, approximately 50 pM ~ approximately 800 pM, approximately 50 pM ~ approximately 750 pM, approximately 50 pM ~ approximately 700 pM, approximately 50 pM M~approx. 650pM, approx. 50pM~approx. 600pM, approx. 50pM~approx. 550pM, approx. 50pM~approx. 500pM, approx. 50pM~approx. 450pM, approx. 50pM~approx. 400pM, approx. 50pM~approx. 350pM, approx. 50pM~approx. 300pM, approx. 50pM~approx. 250pM, approx. 50pM~approx. 200pM M, approximately 50pM to approximately 150pM, approximately 50pM to approximately 100pM, approximately 50pM to approximately 90pM, approximately 50pM to approximately 80pM, approximately 50pM to approximately 70pM, approximately 50pM to approximately 60pM, approximately 60pM to approximately 30nM, approximately 60pM to approximately 25nM, approximately 60pM to approximately 30nM, approximately 60pM to approximately 15nM, approximately 60pM ~ approximately 10nM, approximately 60pM ~ approximately 5nM, approximately 60pM ~ approximately 2nM, approximately 60pM ~ approximately 1nM, approximately 60pM ~ approximately 950pM, approximately 60pM ~ approximately 900pM, approximately 60pM ~ approximately 850pM, approximately 60pM ~ approximately 800pM, approximately 60pM ~ approximately 750pM, approximately 60pM ~ approximately 700pM, approximately 6 0pM~approx. 650pM, approx. 60pM~approx. 600pM, approx. 60pM~approx. 550pM, approx. 60pM~approx. 500pM, approx. 60pM~approx. 450pM, approx. 60pM~approx. 400pM, approx. 60pM~approx. 350pM, approx. 60pM~approx. 300pM, approx. 60pM~approx. 250pM, approx. 60pM~approx. 200pM pM, approximately 60 pM to approximately 150 pM, approximately 60 pM to approximately 100 pM, approximately 60 pM to approximately 90 pM, approximately 60 pM to approximately 80 pM, approximately 60 pM to approximately 70 pM, approximately 70 pM to approximately 30 nM, approximately 70 pM to approximately 25 nM, approximately 70 pM to approximately 30 nM, approximately 70 pM to approximately 15 nM, approximately 70 pM to approximately 10 nM Approximately 70 pM to approximately 5 nm, approximately 70 pM to approximately 2 nm, approximately 70 pM to approximately 1 nm, approximately 70 pM to approximately 950 pM, approximately 70 pM to approximately 900 pM, approximately 70 pM to approximately 850 pM, approximately 70 pM to approximately 800 pM, approximately 70 pM to approximately 750 pM, approximately 70 pM to approximately 700 pM, approximately 70 pM to approximately 650 pM.Approximately 70pM~600pM, approximately 7 0pM~approx. 550pM, approx. 70pM~approx. 500pM, approx. 70pM~approx. 450pM, approx. 70pM~approx. 400pM, approx. 70pM~approx. 350pM, approx. 70pM~approx. 300pM, approx. 70pM~approx. 250pM, approx. 70pM~approx. 200pM, approx. 70pM~approx. 150pM, approx. 70pM~approx. 100pM, approx. 70pM~approx. 90pM, approx. 70pM~approx. 80pM, approx. 80pM~approx. 30nM, approx. 80pM~approx. 25nM, approx. 80pM~approx. 30nM, approx. 80pM~approx. 15nM, approx. 80pM M ~ approximately 10 nmM, approximately 80 pM ~ approximately 5 nmM, approximately 80 pM ~ approximately 2 nmM, approximately 80 pM ~ approximately 1 nmM, approximately 80 pM ~ approximately 950 pM, approximately 80 pM ~ approximately 900 pM, approximately 80 pM ~ approximately 850 pM, approximately 80 pM ~ approximately 800 pM, approximately 80 pM ~ approximately 750 pM, approximately 80 pM ~ approximately 700 pM, approximately 80 pM ~ approximately 650 pM, approximately 80 pM ~ approximately 600 pM, approximately 80 pM ~ approximately 550 pM, approximately 80 pM ~ approximately 500 pM, approximately 80 pM ~ approximately 450 pM, approximately 80 pM ~ approximately 400 pM, approximately 80 pM ~ approximately 3 50pM, approximately 80pM to approximately 300pM, approximately 80pM to approximately 250pM, approximately 80pM to approximately 200pM, approximately 80pM to approximately 150pM, approximately 80pM to approximately 100pM, approximately 80pM to approximately 90pM, approximately 90pM to approximately 30nM, approximately 90pM to approximately 25nM, approximately 90pM to approximately 30nM, approximately 90pM to approximately 15nM, approximately 90pM to approximately 10nM, approximately 90pM to approximately 5nM, approximately 90pM to approximately 2nM, approximately 90pM to approximately 1nM, approximately 90pM to approximately 950pM, approximately 90pM to approximately 900pM, approximately 90 pM ~ approximately 850pM, approximately 90pM ~ approximately 800pM, approximately 90pM ~ approximately 750pM, approximately 90pM ~ approximately 700pM, approximately 90pM ~ approximately 650pM, approximately 90pM ~ approximately 600pM, approximately 90pM ~ approximately 550pM, approximately 90pM ~ approximately 500pM, approximately 90pM ~ approximately 450pM, approximately 90pM ~ approximately 400pM, approximately 90pM ~ approximately 350pM, approximately 90pM ~ approximately 300pM, approximately 90pM ~ approximately 250pM, approximately 90pM ~ approximately 200pM, approximately 90pM ~ approximately 150pM, approximately 90pM ~ approximately 100pM Approximately 100 pM to approximately 30 nm, approximately 100 pM to approximately 25 nm, approximately 100 pM to approximately 30 nm, approximately 100 pM to approximately 15 nm, approximately 100 pM to approximately 10 nm, approximately 100 pM to approximately 5 nm, approximately 100 pM to approximately 2 nm, approximately 100 pM to approximately 1 nm, approximately 100 pM to approximately 950 pM, approximately 100 pM to approximately 90 nm 0pM, approximately 100pM to approximately 850pM, approximately 100pM to approximately 800pM, approximately 100pM to approximately 750pM, approximately 100pM to approximately 700pM, approximately 100pM to approximately 650pM, approximately 100pM to approximately 600pM, approximately 100pM to approximately 550pM, approximately 100pM to approximately 500pM, approximately 100pM to approximately 4 50pM, approximately 100pM to approximately 400pM, approximately 100pM to approximately 350pM, approximately 100pM to approximately 300pM, approximately 100pM to approximately 250pM, approximately 100pM to approximately 200pM, approximately 100pM to approximately 150pM, approximately 150pM to approximately 30nm, approximately 150pM to approximately 25nm, approximately 150pM to approximately 30nm nM, approximately 150pM to approximately 15nM, approximately 150pM to approximately 10nM, approximately 150pM to approximately 5nM, approximately 150pM to approximately 2nM, approximately 150pM to approximately 1nM, approximately 150pM to approximately 950pM, approximately 150pM to approximately 900pM, approximately 150pM to approximately 850pM, approximately 150pM to approximately 800pM, approximately 150 pM~approx. 750pM, approx. 150pM~approx. 700pM, approx. 150pM~approx. 650pM, approx. 150pM~approx. 600pM, approx. 150pM~approx. 550pM, approx. 150pM~approx. 500pM, approx. 150pM~approx. 450pM, approx. 150pM~approx. 400pM, approx. 150pM~approx. 350pM, approx. 15 0pM to approximately 300pM, approximately 150pM to approximately 250pM, approximately 150pM to approximately 200pM, approximately 200pM to approximately 30nM, approximately 200pM to approximately 25nM, approximately 200pM to approximately 30nM, approximately 200pM to approximately 15nM, approximately 200pM to approximately 10nM, approximately 200pM to approximately 5nM, approximately 200pM to approximately 2... nM, approximately 200pM to approximately 1nM, approximately 200pM to approximately 950pM, approximately 200pM to approximately 900pM, approximately 200pM to approximately 850pM, approximately 200pM to approximately 800pM, approximately 200pM to approximately 750pM, approximately 200pM to approximately 700pM, approximately 200pM to approximately 650pM, approximately 200pM to approximately 600pM M, approximately 200pM to approximately 550pM, approximately 200pM to approximately 500pM, approximately 200pM to approximately 450pM, approximately 200pM to approximately 400pM, approximately 200pM to approximately 350pM, approximately 200pM to approximately 300pM, approximately 200pM to approximately 250pM, approximately 300pM to approximately 30nm, approximately 300pM to approximately 25nm.Approximately 300 pM to approximately 30 nm, approximately 300 pM to approximately 15 nm, approximately 300 pM to approximately 10 nm, approximately 300 pM to approximately 5 nm, approximately 300 pM to approximately 2 nm, approximately 300 pM to approximately 1 nm, approximately 300 pM to approximately 950 pM, approximately 300 pM to approximately 900 pM, approximately 300 pM to approximately 850 pM, approximately 300 pM to approximately 800pM, approximately 300pM to approximately 750pM, approximately 300pM to approximately 700pM, approximately 300pM to approximately 650pM, approximately 300pM to approximately 600pM, approximately 300pM to approximately 550pM, approximately 300pM to approximately 500pM, approximately 300pM to approximately 450pM, approximately 300pM to approximately 400pM, approximately 300pM~ Approximately 350 pM, approximately 400 pM to approximately 30 nm, approximately 400 pM to approximately 25 nm, approximately 400 pM to approximately 30 nm, approximately 400 pM to approximately 15 nm, approximately 400 pM to approximately 10 nm, approximately 400 pM to approximately 5 nm, approximately 400 pM to approximately 2 nm, approximately 400 pM to approximately 1 nm, approximately 400 pM to approximately 950 pM, approximately 40 0pM~approx. 900pM, approx. 400pM~approx. 850pM, approx. 400pM~approx. 800pM, approx. 400pM~approx. 750pM, approx. 400pM~approx. 700pM, approx. 400pM~approx. 650pM, approx. 400pM~approx. 600pM, approx. 400pM~approx. 550pM, approx. 400pM~approx. 500pM, approx. 5 00pM ~ approximately 30nM, approximately 500pM ~ approximately 25nM, approximately 500pM ~ approximately 30nM, approximately 500pM ~ approximately 15nM, approximately 500pM ~ approximately 10nM, approximately 500pM ~ approximately 5nM, approximately 500pM ~ approximately 2nM, approximately 500pM ~ approximately 1nM, approximately 500pM ~ approximately 950pM, approximately 500pM ~ approximately 900pM M, approximately 500pM to approximately 850pM, approximately 500pM to approximately 800pM, approximately 500pM to approximately 750pM, approximately 500pM to approximately 700pM, approximately 500pM to approximately 650pM, approximately 500pM to approximately 600pM, approximately 500pM to approximately 550pM, approximately 600pM to approximately 30nm, approximately 600pM to approximately 25nm Approximately 600pM to approximately 30nm, approximately 600pM to approximately 15nm, approximately 600pM to approximately 10nm, approximately 600pM to approximately 5nm, approximately 600pM to approximately 2nm, approximately 600pM to approximately 1nm, approximately 600pM to approximately 950pM, approximately 600pM to approximately 900pM, approximately 600pM to approximately 850pM, approximately 600pM~ Approximately 800 pM, approximately 600 pM to approximately 750 pM, approximately 600 pM to approximately 700 pM, approximately 600 pM to approximately 650 pM, approximately 700 pM to approximately 30 nm, approximately 700 pM to approximately 25 nm, approximately 700 pM to approximately 30 nm, approximately 700 pM to approximately 15 nm, approximately 700 pM to approximately 10 nm, approximately 700 pM to approximately 5 nm.Approximately 700 pM to approximately 2 nm, approximately 700 pM to approximately 1 nm, approximately 700 pM to approximately 950 pM, approximately 700 pM to approximately 900 pM, approximately 700 pM to approximately 850 pM, approximately 700 pM to approximately 800 pM, approximately 700 pM to approximately 750 pM, approximately 800 pM to approximately 30 nm, approximately 800 pM to approximately 25 nm, approximately 800 pM to approximately 30 nm, approximately 800 pM to approximately 15 nm, approximately 800 pM to approximately 10 nm, approximately 800 pM to approximately 5 nm, approximately 800 pM to approximately 2nM, approximately 800pM to approximately 1nM, approximately 800pM to approximately 950pM, approximately 800pM to approximately 900pM, approximately 800pM to approximately 850pM, approximately 900pM to approximately 30nM, approximately 900pM to approximately 25nM, approximately 900pM to approximately 30nM, approximately 900pM to approximately 15nM, approximately 900pM to approximately 10nM, approximately 900pM to approximately 5nM, approximately 900pM to approximately 2nM, approximately 900pM to approximately 1nM, approximately 900pM to approximately 950pM, approximately 1nM to approximately 30nM, approximately 1nM to approximately 25nM, approximately 1nM to approximately 20nM, approximately 1nM to approximately 15nM, approximately 1nM to approximately 10nM, approximately 1nM to approximately 5nM, approximately 2nM to approximately 30nM, approximately 2nM to approximately 25nM, approximately 2nM to approximately 20nM, approximately 2nM to approximately 15nM, approximately 2nM to approximately 10nM, approximately 2nM to approximately 5nM, approximately 4nM to approximately 30nM, approximately 4nM to approximately 25nM, approximately 4nM to approximately 20nM, approximately 4nM to approximately 15nM, approximately 4nM to approximately 10nM Approximately 4nM to 5nM, approximately 5nM to 30nM, approximately 5nM to 25nM, approximately 5nM to 20nM, approximately 5nM to 15nM, approximately 5nM to 10nM, approximately 10nM to 30nM, approximately 10nM to 25nM, approximately 10nM to 20nM, approximately 10nM to 15nM, approximately 15nM to 30nM, approximately 15nM to 25nM, approximately 15nM to 20nM, approximately 20nM to 30nM, approximately 20nM to 30nM, approximately 20nM to 25nM) of K, D The combination of the でその standard and the することができる.

[0067] Any of the target binding domains described herein may be administered at a concentration of about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM). M, about 2nM to about 4nM, about 2nM to about 3nM, about 3nM to about 10nM, about 3nM to about 9nM, about 3nM to about 8nM, about 3nM to about 7nM, about 3nM to about 6nM, about 3nM to about 5nM, about 3nM to about 4 nM, about 4nM to about 10nM, about 4nM to about 9nM, about 4nM to about 8nM, about 4nM to about 7nM, about 4nM to about 6nM, about 4nM to about 5nM, about 5nM to about 10nM, about 5nM to about 9nM, about 5nM K of about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to about 10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM D can bind to its target.

[0068] A variety of different methods known in the art can be used to quantify any of the antigen binding protein constructs described herein. D Values ​​can also be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecular binding kinetic assays, etc.).

[0069] antigen-binding domain In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0070] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0071] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are antigen-binding domains. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each antigen-binding domains.

[0072] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is an scFv or a single-domain antibody (e.g., a VHH or VNAR domain).

[0073] In some examples, the antigen binding domain (e.g., any of the antigen binding domains described herein) is selected from the group consisting of CD16a (see, e.g., as described in U.S. Pat. No. 9,035,026), CD28 (see, e.g., as described in U.S. Pat. No. 7,723,482), CD3 (see, e.g., as described in U.S. Pat. No. 9,226,962), CD33 (see, e.g., as described in U.S. Pat. No. 8,759,494), CD20 (see, e.g., as described in WO2014 / 026054), CD19 (see, e.g., as described in U.S. Pat. No. 9,701,758), CD22 (see, e.g., as described in WO2003 / 104425), and the like. No. 2012 / 0171197), IL-10 (see, e.g., U.S. Patent Application Publication No. 2016 / 0340413), PDL-1 (see, e.g., Drees, et al., Protein Express. Purif. 94:60-66, 2014), TIGIT (see, e.g., U.S. Patent Application Publication No. 2017 / 0198042), PD-1 (see, e.g., U.S. Pat. Appl. Pub. No. 2017 / 0198042), No. 7,488,802), TIM3 (see, e.g., U.S. Pat. No. 8,552,156), CTLA4 (see, e.g., WO2012 / 120125), MICA (see, e.g., WO2016 / 154585), MICB (see, e.g., U.S. Pat. No. 8,753,640), IL-6 (see, e.g., Gejima et al., Human Antibodies 11(4):121-129, 2002), IL-8 (see, e.g., U.S. Pat. No. 6,117,980), TNFα (see, e.g., Geng et al., Human Antibodies 11(4):121-129, 2002), TNFα (see, e.g., U.S. Pat. No. 6,117,980 ... al., Immunol. Res. 62(3):377-385, 2015), CD26a (see, e.g., WO2017 / 189526), ​​CD36 (see, e.g., U.S. Patent Application Publication No. 2015 / 0259429), ULBP2 (see, e.g., U.S. Patent No. 9,273,136), CD30 (see, e.g., Homach et al., Immunol. Res. 62(3):377-385, 2015), CD26b (see, e.g., WO2017 / 189526), ​​CD36 (see, e.g., U.S. Patent Application Publication No. 2015 / 0259429), ULBP2 (see, e.g., U.S. Patent No. 9,273,136), CD3 al., Scand. J. Immunol. 48(5):497-501, 1998), CD200 (see, e.g., U.S. Pat. No. 9,085,623), IGF-1R (see, e.g., U.S. Patent Application Publication No. 2017 / 0051063), MUC4AC (see, e.g., WO2012 / 170470), MUC5AC (see, e.g., U.S. Pat. No. 9,238,084), Trop-2 (see, e.g., WO2013 / 068946), CMET (see, e.g., Edwardraja et al., Biotechnol. Bioeng. 106(3):367-375, 2010), EGFR (see, e.g., Akbari et al., Protein Expr. Purif. 127:8-15, 2016), HER1 (see, for example, those described in U.S. Patent Application Publication No. 2013 / 0274446), HER2 (see, for example, those described in Cao et al., Biotechnol. Lett.37(7):1347-1354, 2015), HER3 (see, e.g., U.S. Pat. No. 9,505,843), PSMA (see, e.g., Parker et al., Protein Expr. Purif. 89(2):136-145, 2013), CEA (see, e.g., WO1995 / 015341), B7H3 (see, e.g., U.S. Pat. No. 9,371,395), EPCAM (see, e.g., WO2014 / 159531), BCMA (see, e.g., Smith et al., al., Mol. Ther. 26(6):1447-1456, 2018), P-cadherin (see, e.g., U.S. Pat. No. 7,452,537), CEACAM5 (see, e.g., U.S. Pat. No. 9,617,345), UL16 binding protein (see, e.g., WO2017 / 083612), HLA-DR (see, e.g., Pistillo et al., Mol. Ther. 26(6):1447-1456, 2018), and the like. al., Exp. Clin. Immunogenet. 14(2):123-130, 1997), DLL4 (see, e.g., those described in WO2014 / 007513), TYRO3 (see, e.g., those described in WO2016 / 166348), AXL (see, e.g., those described in WO2012 / 175692), MER (see, e.g., those described in WO2016 / 106221), CD122 (see, e.g., those described in U.S. Patent Application Publication No. 2016 / 0367664), CD155 (see, e.g., those described in WO2017 / 149538), or PDGF-DD (see, e.g., those described in U.S. Patent No. 9,441,034).

[0074] Each antigen-binding domain present in any of the multi-chain chimeric polypeptides described herein is independently selected from the group consisting of a VHH domain, a VNAR domain, and an scFv. In some embodiments, any of the antigen-binding domains described herein is selected from the group consisting of BiTe, (scFv)2, nanobody, nanobody-HSA, DART, Ta ndAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. Additional examples of antigen-binding domains that can be used in any of the multi-chain chimeric polypeptides are known in the art.

[0075] VHH domains are single monomeric variable antibody domains that can be found in camelids. VNAR domains are single monomeric variable antibody domains that can be found in cartilaginous fish. VHH domains and V NARNon-limiting examples of domains are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016, De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006, De Meyer et al., Trends Biotechnol. 32:263-270, 2014, Kijanka et al., Nanomedicine 10:161-174, 2015, Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014, Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016, Mujic-Delic et al., Trends Pharmacol.Sci.35:247-255,2014, Muyldermans,J.Biotechnol.74:277-302,2001, Muyldermans et al.,Trends Biochem.Sci.26:230-235,2001, Muyldermans,Ann.Rev.Biochem.82:775-797,2013, Rahbarizadeh et al.,Immunol.Invest.40:299-338,2011, Van Audenhove et al.,EBioMedicine 8:40-48,2016, Van Bockstaele et al.,Curr.Opin.Investig.Drugs 10:1212-1224,2009, Vincke et al.,Methods Mol. Biol. 911:15-26, 2012, and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0076] In some embodiments, each of the antigen-binding domains in a multi-chain chimeric polypeptide described herein are both VHH domains, or at least one antigen-binding domain is a VHH domain. In some embodiments, each of the antigen-binding domains in a multi-chain chimeric polypeptide described herein are both VNAR domains, or at least one antigen-binding domain is a VNAR domain. In some embodiments, each of the antigen-binding domains in a multi-chain chimeric polypeptide described herein are both scFv domains, or at least one antigen-binding domain is an scFv domain.

[0077] In some embodiments, two or more polypeptides present in the multi-chain chimeric polypeptide are assembled (e.g., non-covalently assembled) to form any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), VHH-scAb, VHH-Fab, double-scFab, F(ab'), diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-hole common light chain, knobs-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Minibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv , F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scdiabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock-and-lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv1-PEG-scFv2 can be formed. For a description of these elements, see, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, which is incorporated herein in its entirety. Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments. Additional examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA, e.g., human or humanized IgA1 or IgA2), antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD), antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE), or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0078] An "Fv" fragment comprises a non-covalent dimer of one heavy-chain and one light-chain variable domain.

[0079] A "Fab" fragment contains the heavy and light chain variable domains of an Fv fragment as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ) is included.

[0080] An "F(ab')2" fragment contains two Fab fragments linked by a disulfide bond near the hinge region.

[0081] "Dual variable domain immunoglobulins" or "DVD-Ig" are defined, for example, as described in DiGiammarino et al., Methods, 2002, pp. 111-114, 2002, each of which is incorporated by reference in its entirety. Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Patent Nos. 7,612,181, 8,258,268, 8,586,714, 8,716,450, 8,722,855, 8,735,546, and 8,822,645.

[0082] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0083] In some embodiments of any of the antigen-binding domains described herein, they are capable of binding to an antigen selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

[0084] Additional examples and embodiments of antigen binding domains are known in the art.

[0085] Soluble interleukin or cytokine proteins In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain can be a soluble interleukin or soluble cytokine protein. In some embodiments, the soluble interleukin or soluble cytokine protein is selected from the group consisting of IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L are provided below. Human soluble IL-2 (SEQ ID NO: 9) aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr witfcqsiis tlt Human soluble IL-3 (SEQ ID NO: 10) apmtqttplkt swvncsnmid eiithlkqpp lplldfnnln gedqdilmen nlrrpnleaf nravkslqna saiesilknl lpclplataa ptrhpihikd gdwnefrrkl tfylktlena qaqqttlsla if Human soluble IL-7 (SEQ ID NO: 11) dcdiegkdgkqyesv lmvsidqlld smkeigsncl nnefnffkrh icdankegmf lfraarklrq flkmnstgdf dlhllkvseg ttillnctgq vkgrkpaalg eaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki lmgtkeh Human soluble IL-8 (SEQ ID NO: 12) egavlprsak elrcqcikty skpfhpkfik elrviesgph canteiivkl sdgrelcldp kenwvqrvve kflkraens Human soluble IL-10 (SEQ ID NO: 13) spgqgtqsensc thfpgnlpnm lrdlrdafsr vktffqmkdq ldnlllkesl ledfkgylgc qalsemiqfy leevmpqaen qdpdikahvn slgenlktlr lrlrrchrfl pcenkskave qvknafnklq ekgiykamse fdifinyiea ymtmkirn Human soluble IL-15 (SEQ ID NO: 14) Nwvnvisdlkki edliqsmhid atlytesdvh psckvtamkc flllelqvisl esgdasihdt venliilann slssngnvte sgckeceele eknikeflqs fvhivqmfin ts Human soluble IL-17 (SEQ ID NO: 15) gitiprn pgcpnsedkn fprtvmvnln ihnrntntnp krssdyynrs tspwnlhrne dperypsviw eakcrhlgci nadgnvdyhm nsvpiqqeil vlrrepphcp nsfrlekilv svgctcvtpi vhhva Human soluble IL-18 (SEQ ID NO: 16) yfgklesklsvirn lndqvlfidq gnrplfedmt dsdcrdnapr tifiismykd sqprgmavti svkcekistl scenkiisfk emnppdnikd tksdiiffqr svpghdnkmq fesssyegyf lacekerdlf klilkkedel gdrsimftvq ned Human soluble PDGF-DD (SEQ ID NO: 17) rdtsatpqsasi kalrnanlrr desnhltdly rrdetiqvkg ngyvqsprfp nsyprnlllt wrlhsqentr iqlvfdnqfg leeaeendicr ydfvevedis etstiirgrw cghkevppri ksrtnqikit fksddyf vak pgfkiyysll edfqpaaase tnwesvtssi sgvsynspsv tdptliadal dkkiaefdtv edllkyfnpe swqedlenmy ldtpryrgrs yhdrkskvdl drlnddakry sctprnysvn ireelklanv vffprcllvq rcggncgcgt vnwrsctcns gktvkkyhev lqfepghikr rgraktmalv diqldhherc dcicssrppr Human soluble SCF (SEQ ID NO: 18) egicrnrvtnnvkdv tklvanlpkd ymitlkyvpg mdvlpshcwi semvvqlsds ltdlldkfsn iseglsnysi idklvnivdd lvecvkenss kdlkksfksp eprlftpeef frifnrsida fkdfvvaset sdcvvsstls pekdsrvsvt kpfmlppvaa sslrndssss nrkaknppgd sslhwaamal palfsliigf afgalywkkr qpsltraven iqineednei smlqekeref qev Human soluble FLT3L (SEQ ID NO: 19) tqdcsfqhspissd favkirelsd yllqdypvtv asnlqdeelc gglwrlvlaq rwmerlktva gskmqgller vnteihfvtk cafqpppscl rfvqtnisrl lqetseqlva lkpwitrqnf srclelqcqp dsstlpppws prpleatapt apqppllllll llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl llveh

[0086] Non-limiting examples of soluble MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 are provided below. Human soluble MICA (SEQ ID NO: 20) ephslry nltvlswdgs vqsgfltevh ldgqpflrcd rqkcrakpqg qwaedvlgnk twdretrdlt gngkdlrmtl ahikdqkegl hslqeirvce ihednstrss qhfyydgelf lsqnletkew tmpqssraqt lamnvrnflk edamktkthy hamhadclqe lrrylksgvv lrrtvppmvn vtrseasegn itvtcrasgf ypwnitlswr qdgvslshdt qqwgdvlpdg ngtyqtwvat ricqgeeqrf tcymehsgnh sthpvpsgkv lvlqshwqtf hvsavaaaai fviiifyvrc ckkktsaaeg pelvslqvld qhpvgtsdhr datqlgfqpl msdlgstgst ega Human soluble MICB (SEQ ID NO: 21) aephslry nlmvlsqdes vqsgflaegh ldgqpflryd rqkrrakpqg qwaedvlgak twdtetedlt engqdlrrtl thikdqkggl hslqeirvce ihedsstrgs rhfyydgelf lsqnletqes tvpqssraqt lamnvtnfwk edamktkthy ramqadclqk lqrylksgva irrtvppmvn vtcsevsegn itvtcrassf yprnitltwr qdgvslshnt qqwgdvlpdg ngtyqtwvat rirqgeeqrf tcymehsgnh gthpvpsgkv lvlqsqrtdf pyvsaampcf viiilcvpc ckkktsaaeg pelvslqvld qhpvgtgdhr daaqlgfqpl msatgstgst ega Human soluble ULBP1 (SEQ ID NO: 22) wvdthclcydfiit pksrpepqwc evqglvderp flhydcvnhk akafaslgkk vnvtktweeq tetlrdvvdf lkgqlldiqv enlipieplt lqarmscehe ahghgrgswq flfngqkfll fdsnnrkwta lhpgakkmte kweknrdvtm ffqkislgdc kmwleeflmy weqmldptkp pslapg Human soluble ULBP2 (SEQ ID NO: 23) gradphslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt vtpvsplgkk lnvttawkaq npvlrevvdi lteqlrdiql enytpkeplt lqarmsceqk aeghssgswq fsfdgqifll fdsekrmwtt vhpgarkmke kwendkvvam sfhyfsmgdc igwledflmg mdstlepsag aplams Human soluble ULBP3 (SEQ ID NO: 24) dahslwynfti ihlprhgqqw cevqsqvdqk nflsydcgsd kvlsmghlee qlyatdawgk qlemlrevgq rlrleladte ledftpsgpl tlqvrmscec eadgyirgsw qfsfdgrkfl lfdsnnrkwt vvhagarrmk ekwekdsglt tffkmvsmrd ckswlrdflm hrkkrlepta pptmapg human soluble ULBP4 (SEQ ID NO: 25) hslcfnftik slsrpgqpwc eaqvflnknl flqynsdnnm vkplgllgkk vyatstwgel tqtlgevgrd lrmllcdikp qiktsdpstl qvemfcqrea erctgaswqf atngeksllf damnmtwtvi nheaskiket wkkdrgleky frklskgdcd hwlreflghw eampeptvsp vnasdihwss sslpdrwiil gafillvlmg ivlicvwwqn gewqaglwpl rts Human soluble ULBP5 (SEQ ID NO: 26) gladp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgskt vtpvsplgkk lnvttawkaq npvlrevvdi lteqlldiql enyipkeplt lqarmsceqk aeghgsgswq lsfdgqifll fdsenrmwtt vhpgarkmke kwendkdmtm sfhyismgdc tgwledflmg mdstlepsag apptmssg Human soluble ULBP6 (SEQ ID NO: 27) rrddp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt vtpvsplgkk lnvtmawkaq npvlrevvdi lteqlldiql enytpkeplt lqarmsceqk aeghssgswq fsidgqtfll fdsekrmwtt vhpgarkmke kwendkdvam sfhyismgdc igwledflmg mdstlepsag aplamssg

[0087] Additional examples of soluble interleukin and cytokine proteins are known in the art.

[0088] Soluble receptors In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are soluble iodoforms. In some embodiments, the soluble receptor is a soluble TGF-β receptor II (TGF-β RII) (see, e.g., Yung et al., Am. J. Resp. Crit. Care Med. 194(9):1140-1151, 2016), a soluble TGF-β RIII (see, e.g., Heng et al., Placenta 57:320, 2017), a soluble NKG2D (see, e.g., Cosman et al., Immunity 14(2):123-133, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp30 (see, e.g., Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp44 (see, e.g., those described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp46 (see, e.g., those described in Mandelboim et al., Nature 409:1055-1060, 2001, Costa et al., Nature 409:1055-1060, 2001, Costa et al., Nature 409:...

Claims

1. (a)(i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of a pair of affinity domains; and a first chimeric polypeptide comprising: (b)(i) a second domain of the pair of affinity domains; (ii) a second target binding domain; and a second chimeric polypeptide comprising wherein the first chimeric polypeptide and the second chimeric polypeptide are associated via binding between the first domain and the second domain of the pair of affinity domains.

2. The multi-chain chimeric polypeptide of claim 1 , wherein the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide.

3. The multi-chain chimeric polypeptide of claim 1, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

4. The multi-chain chimeric polypeptide of any one of claims 1 to 3, wherein the soluble tissue factor domain and the first domain of the pair of affinity domains are immediately adjacent to each other within the first chimeric polypeptide.

5. The multi-chain chimeric polypeptide of any one of claims 1 to 3, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain of the pair of affinity domains.

6. The multi-chain chimeric polypeptide of any one of claims 1 to 5, wherein the second domain and the second target binding domain of the pair of affinity domains are immediately adjacent to each other within the second chimeric polypeptide.

7. The multi-chain chimeric polypeptide of any one of claims 1 to 5, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain.

8. The multi-chain chimeric polypeptide of any one of claims 1 to 7, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

9. The multi-chain chimeric polypeptide of any one of claims 1 to 7, wherein the first target binding domain and the second target binding domain specifically bind to different antigens.

10. The multi-chain chimeric polypeptide of any one of claims 1 to 9, wherein one or both of the first target-binding domain and the second target-binding domain is an antigen-binding domain.

11. One or both of the first target binding domain and the second target binding domain may bind to any of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL- 8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI liga 11. The multi-chain chimeric polypeptide of any one of claims 1 to 10, which specifically binds to a target selected from the group consisting of a scMHCII ligand, a scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

12. The multi-chain chimeric polypeptide of any one of claims 1 to 10, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein.

13. 13. The multi-chain chimeric polypeptide of claim 12, wherein the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

14. The multi-chain chimeric polypeptide of any one of claims 1 to 10, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor.

15. The soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NK 15. The multichain chimeric polypeptide of claim 14, which is p46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28.

16. The multi-chain chimeric polypeptide of any one of claims 1 to 15, wherein the first chimeric polypeptide further comprises one or more additional target binding domain(s).

17. The multi-chain chimeric polypeptide of any one of claims 1 to 16, wherein the second chimeric polypeptide further comprises one or more additional target binding domains.

18. The multi-chain chimeric polypeptide of any one of claims 1 to 17, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

19. 19. The multi-chain chimeric polypeptide of claim 18, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:

1.

20. The multi-chain chimeric polypeptide of any one of claims 1 to 19, wherein the pair of affinity domains are a sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15.

21. 20. The multi-chain chimeric polypeptide of any one of claims 1 to 19, wherein the pair of affinity domains is selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter / docking tag modules based on mutant RNase I fragments, and SNARE modules based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

22. The multi-chain chimeric polypeptide of any one of claims 1 to 21, wherein the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

23. A composition comprising any of the multi-chain chimeric polypeptides of claims 1 to 22.

24. 24. The composition of claim 23, which is a pharmaceutical composition.

25. 25. A kit comprising at least one dose of the composition of claim 23 or 24.

26. 1. A method for stimulating immune cells, comprising: contacting immune cells with an effective amount of any of the multi-chain chimeric polypeptides of claims 1 to 22 or the composition of claims 23 or 24. A method comprising:

27. 1. A method for inducing or increasing immune cell proliferation, comprising: contacting immune cells with an effective amount of any of the multi-chain chimeric polypeptides of claims 1 to 22 or the composition of claims 23 or 24. A method comprising:

28. 1. A method for inducing differentiation of immune cells into memory or memory-like immune cells, comprising: contacting immune cells with an effective amount of any of the multi-chain chimeric polypeptides of claims 1 to 22 or the composition of claims 23 or 24. A method comprising:

29. The method of any one of claims 26 to 28, wherein the immune cells are contacted in vitro.

30. The method of any one of claims 26 to 28, wherein the immune cells are contacted in vivo.

31. 31. The method of any one of claims 26 to 30, wherein the immune cells are selected from the group consisting of immature thymocytes, peripheral blood lymphocytes, naive T cells, multipotent Th cell precursors, lymphoid progenitor cells, Treg cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8+ T cells, CD4+ T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells.

32. The method of any one of claims 26 to 31, wherein the immune cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor.

33. 25. A method for killing cancer cells, infected cells, or senescent cells in a subject in need thereof, comprising administering to the subject any of the multi-chain chimeric polypeptides of claims 1 to 22 or the composition of claim 23 or 24. The method comprises administering a therapeutically effective amount of

34. 25. A method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides of claims 1-22 or the composition of claim 23 or 24.

35. 35. The method of claim 33 or 34, wherein the subject has been identified or diagnosed as having cancer, an age-related disease or condition, or an infectious disease.

36. 36. The method of claim 35, wherein the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

37. 36. The method of claim 35, wherein the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

38. 36. The method of claim 35, wherein the infectious disease is infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

39. A nucleic acid encoding any of the multi-chain chimeric polypeptides of any one of claims 1 to 22.

40. A vector comprising the nucleic acid of claim 39.

41. 41. A cell comprising the nucleic acid of claim 39 or the vector of claim 40.

42. 1. A method for producing a multi-chain chimeric polypeptide, comprising: Culturing the cell of claim 41 in a culture medium under conditions sufficient to result in the production of said multi-chain chimeric polypeptide; and recovering said multi-chain chimeric polypeptide from said cells and / or said culture medium.

43. 43. A multi-chain chimeric polypeptide produced by the method of claim 42.

44. 20. The multi-chain chimeric polypeptide of claim 18, wherein the human soluble tissue factor domain does not initiate blood clotting.

45. The multi-chain chimeric polypeptide of any one of claims 1 to 22, wherein the soluble tissue factor domain comprises or consists of a sequence derived from wild-type soluble human tissue factor.

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