Single-chain chimeric polypeptides and uses thereof

Abstract

Chimeric polypeptides comprising a soluble tissue factor and an antigen binding domain are provided.SOLUTION: Provided herein are single-chain chimeric polypeptides comprising (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a second target binding domain. Also provided herein are methods of using these single-chain chimeric polypeptides and nucleic acids encoding these single-chain chimeric polypeptides.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 725,038, filed August 30, 2018, U.S. Patent Application No. 62 / 817,244, filed March 12, 2019, U.S. Patent Application No. 62 / 746,832, filed October 17, 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 / 881,039, filed July 31, 2019, each of which is incorporated herein by reference in its entirety.

[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 a 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 the rapid cleavage of FX to FXa and FIX to FIXa. The resulting proteolytic activity of FXa and the activated membrane surface then inefficiently converts small amounts of prothrombin to thrombin. Thrombin generated by FXa initiates platelet activation, activating trace amounts of pro-cofactors 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 rapidly generates FXa. FXa complexes with FVa to form the prothrombinase complex on the activated platelet surface, which rapidly cleaves 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 the cell surface tissue factor-FVIIa complex. Although PS exposure is known to reduce the apparent Km for activation of FIX and FX by the tissue factor-FVIIa complex, additional mechanisms may involve conformational rearrangements of tissue factor or tissue factor-FVIIa and subsequent exposure of substrate-binding sites. 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 an antigen-binding domain. Based on this discovery, single-chain chimeric polypeptides comprising (i) a first target-binding domain, (ii) a soluble tissue factor domain, and (iii) a second target-binding domain are provided herein. Also provided herein are compositions comprising any of the single-chain chimeric polypeptides described herein, nucleic acids encoding any of the single-chain chimeric polypeptides described herein, and cells comprising any of the nucleic acids encoding any of the single-chain chimeric polypeptides described herein. Also provided herein are methods for stimulating immune cells and treating subjects in need of treatment, including the use of any of the single-chain chimeric polypeptides described herein. Also provided herein are methods for producing any of the single-chain chimeric polypeptides described herein. Additional uses of any of the single-chain chimeric polypeptides are described herein.

[0007] therefore, (i) a first target binding domain; (ii) a soluble tissue factor domain, and (iii) a second target-binding domain Provided herein are single-chain chimeric polypeptides comprising: In some embodiments, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain. In some embodiments, the soluble tissue factor domain and the second target binding domain are immediately adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the second target binding domain. In some embodiments, the first target binding domain and the second target binding domain are immediately adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain. In some embodiments, the second target binding domain and the soluble tissue factor domain are immediately adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the second target binding domain and the soluble tissue factor domain. In some embodiments, 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-D D, binds to a target selected from the group consisting of a TGF-β receptor II (TGF-βRII) ligand, 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. In some embodiments, one or both of the first target binding domain and the second target binding domain is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein. In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding protein.In some embodiments, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some embodiments, the soluble interleukin receptor, the soluble cytokine receptor, or the soluble cell surface receptor is a soluble TGF-β receptor II (TGF-βRII), a soluble TGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28. 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:9. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the single-chain chimeric polypeptide of 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.In some embodiments, the soluble human tissue factor domain does not contain any of the following: a lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, an isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, a tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, an aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, a tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, an arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and a 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 single-chain chimeric polypeptide does not stimulate blood clotting in a mammal. In some embodiments, the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and / or C-terminus. In some embodiments, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus. In some embodiments, the one or more additional target binding domains are immediately adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its C-terminus. In some embodiments, one of the one or more additional target binding domains is immediately adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain.In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domain and the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus and C-terminus. In some embodiments, one of the one or more additional antigen binding domains at the N-terminus is immediately adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen binding domains at the N-terminus and the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments, one of the one or more additional antigen binding domains at the C-terminus is immediately adjacent to the first target binding domain, the second target binding domain, or the soluble tissue factor domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional antigen-binding domains at the C-terminus and the first target binding domain, the second target binding domain, or the soluble tissue factor domain. 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 are antigen binding domains. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each antigen binding domains. In some embodiments, the antigen binding domain comprises an scFv or a single domain antibody. 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. H ER3, 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, scMHCII ligand, scTCR ligand, IL 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 a target selected from the group consisting of IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-D receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 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 interleukin or cytokine proteins. In some embodiments, the soluble interleukin or cytokine protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding protein. 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 NKp 44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.In some embodiments, the single-chain chimeric polypeptide further comprises a signal sequence at its N-terminus. In some embodiments, the single-chain chimeric polypeptide further comprises a peptide tag positioned at the N-terminus or C-terminus of the single-chain chimeric polypeptide.

[0008] Also provided herein are compositions comprising any of the above-described single-chain chimeric polypeptides. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the single-chain chimeric polypeptide comprises at least one dose of the composition in the kit.

[0009] Also provided herein are methods of stimulating immune cells, the methods comprising contacting immune cells with an effective amount of any of the above-described single-chain chimeric polypeptides or compositions. In some embodiments, the methods comprise contacting the immune cells in vitro. In some embodiments, the methods comprise obtaining the immune cells from a subject. In some embodiments, the methods further comprise obtaining the immune cells from a subject prior to the contacting step. In some embodiments, the methods comprise 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, 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 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 previously 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 decrease in lung tissue elasticity, 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 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 cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.In some embodiments, the method includes diagnosing or identifying the subject as having an infectious disease, in some embodiments, 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.

[0010] Also provided herein are methods of inducing or increasing the proliferation of immune cells, the methods comprising contacting immune cells with an effective amount of any of the single-chain chimeric polypeptides or compositions described herein. In some embodiments, the methods comprise contacting the immune cells in vitro. In some embodiments, the methods comprise obtaining the immune cells from a subject. In some embodiments, the methods further comprise obtaining the immune cells from a subject prior to the contacting step. In some embodiments, the methods comprise 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, 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 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 previously 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 decrease in lung tissue elasticity, 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 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 cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.In some embodiments, the method includes diagnosing or identifying the subject as having an infectious disease, in some embodiments, 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.

[0011] Also provided herein are methods of inducing differentiation of immune cells into memory or memory-like immune cells, the methods comprising contacting immune cells with an effective amount of any of the above-described single-chain chimeric polypeptides or compositions. In some embodiments, the methods comprise contacting the immune cells in vitro. In some embodiments, the methods comprise obtaining the immune cells from a subject. In some embodiments, the methods further comprise obtaining the immune cells from a subject prior to the contacting step. In some embodiments, the methods comprise 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+ 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 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 decrease in lung tissue elasticity, 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 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 cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, 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.

[0012] 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 above-described single-chain chimeric polypeptides or compositions. 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 comprises identifying or diagnosing a 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, 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, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

[0013] Also provided herein are methods of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the above-described single-chain chimeric polypeptides or compositions. 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 cancer, lung cancer, renal cell carcinoma, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the method includes 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 decrease in lung tissue elasticity, 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 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 single-chain chimeric polypeptides. Some embodiments include vectors comprising the above-described nucleic acids. In some embodiments, the vectors are expression vectors. Some embodiments include cells comprising the above-described nucleic acids or vectors.

[0015] Also provided herein are methods for producing a single-chain chimeric polypeptide, comprising culturing cells in a culture medium under conditions sufficient to result in the production of the single-chain chimeric polypeptide, and recovering the single-chain chimeric polypeptide from the cells and / or the culture medium. Some embodiments include a single-chain chimeric polypeptide produced by the above-described method. In some embodiments of any of the single-chain chimeric polypeptides provided herein, the human soluble tissue factor domain does not initiate blood coagulation. 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.

[0016] In some embodiments, the variant soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO: 96. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO: 96. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO: 96. In some embodiments, the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO: 96. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO: 97. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 90% identical to SEQ ID NO: 97. In some embodiments, the soluble human tissue factor domain comprises a sequence at least 95% identical to SEQ ID NO: 97. In some embodiments, the soluble human tissue factor domain comprises a sequence 100% identical to SEQ ID NO: 97.

[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 in the broadest sense herein 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 its 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 its 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 specified, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between a member of the antigen-binding domain and the 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 "single-chain polypeptide" refers to a single protein chain.

[0027] The term "affinity domain pair" refers to a -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) DA pair of affinity domains is a pair of protein domains that specifically bind to each other at a specific site. In some examples, the pair of affinity domains can be a pair of naturally occurring proteins. In some embodiments, the pair of affinity domains can be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs 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 induce cell death or cell stop of mammalian pathogenic cells (e.g., cancer cells). 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 improving at least one symptom of a disorder. In some examples, the disorder being treated is cancer, and improving at least one symptom of cancer includes reducing abnormal proliferation, gene expression, signal transduction, translation, and / or secretion of a factor. Generally, treatment methods involve administering to a subject in need of treatment, or a subject 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 those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art may also be used.The materials, methods, and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In the event of any conflict, the present specification, including definitions, shall control.

[0032] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [The present invention 1001] (i) a first target binding domain; (ii) a soluble tissue factor domain, and (iii) a second target-binding domain A single-chain chimeric polypeptide comprising: [The present invention 1002] 1001. The single-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. [The present invention 1003] 1001. The single-chain chimeric polypeptide of the present invention, further comprising a linker sequence between said first target binding domain and said soluble tissue factor domain. [The present invention 1004] The single-chain chimeric polypeptide of any of claims 1001 to 1003, wherein the soluble tissue factor domain and the second target binding domain are immediately adjacent to each other. [The present invention 1005] The single-chain chimeric polypeptide of any of claims 1001 to 1003, further comprising a linker sequence between the soluble tissue factor domain and the second target-binding domain. [The present invention 1006] The single-chain chimeric polypeptide of any of claims 1001 to 1005, wherein the first target-binding domain and the second target-binding domain specifically bind to the same antigen. [The present invention 1007] 1006. The single-chain chimeric polypeptide of the present invention, wherein said first target-binding domain and said second target-binding domain specifically bind to the same epitope. [The present invention 1008] 1007. The single-chain chimeric polypeptide of the present invention, wherein said first target binding domain and said second target binding domain comprise the same amino acid sequence. [The present invention 1009] The single-chain chimeric polypeptide of any of claims 1001 to 1005, wherein the first target-binding domain and the second target-binding domain specifically bind to different antigens. [The present invention 1010] The single-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 ULBP. 2, 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, PDGFDD, TGF-β receptor II (TGF-βR II) A single-chain chimeric polypeptide of any of claims 1001 to 1010, which binds to a target selected from the group consisting of a TGF-βRIII ligand, a DNAM1 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 single-chain chimeric polypeptide of any of claims 1001 to 1010, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein. [The present invention 1013] The single-chain chimeric polypeptide of the present invention, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-D, SCF, FLT3L, MICA, MICB, and ULP16 binding protein. [The present invention 1014] The single-chain chimeric polypeptide of any of claims 1001 to 1010, wherein one or both of the first target-binding domain and the second target-binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. [The present invention 1015] The single-chain chimeric polypeptide of the present invention 1014, wherein the soluble interleukin receptor, the soluble cytokine receptor, or the soluble cell surface 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. [The present invention 1016] The single-chain chimeric polypeptide of any of claims 1001 to 1015, wherein the soluble tissue factor domain is a soluble human tissue factor domain. [The present invention 1017] 1016. The single-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:9. [The present invention 1018] 10. The single-chain chimeric polypeptide of any of claims 1001 to 1017, further comprising one or more additional target binding domains at its N-terminus and / or C-terminus. [The present invention 1019] A composition comprising any one of the single-chain chimeric polypeptides of the present invention 1001 to 1018. [The present invention 1020] The composition of the present invention 1019, which is a pharmaceutical composition. [The present invention 1021] A kit comprising at least one dose of the composition of the present invention 1019 or 1020. [The present invention 1022] 1. A method for stimulating immune cells, comprising: A method comprising contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides of the present invention 1001 to 1018 or the composition of the present invention 1019 or 1020. [The present invention 1023] 1. A method for inducing or increasing immune cell proliferation, comprising: A method comprising contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides of the present invention 1001 to 1018 or the composition of the present invention 1019 or 1020. [The present invention 1024] 1. A method for inducing differentiation of immune cells into memory or memory-like immune cells, comprising: A method comprising contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides of the present invention 1001 to 1018 or the composition of the present invention 1019 or 1020. [The present invention 1025] The method of any one of claims 1022 to 1024, wherein the immune cells are contacted in vitro. [The present invention 1026] The method of any one of claims 1022 to 1024, wherein the immune cells are contacted in vivo. [The present invention 1027] 1027. The method of any one of claims 1022 to 1026, wherein the immune cells are selected from the group consisting of immature thymocytes, peripheral blood lymphocytes, naive T cells, pluripotent 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 1028] 1028. The method of any of claims 1022 to 1027, wherein said immune cells have previously been genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor. [The present invention 1029] The method of any of claims 1022 to 1028, further comprising administering the immune cells to a subject in need thereof. [The present invention 1030] 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 single-chain chimeric polypeptides of the present inventions 1001 to 1018 or the composition of the present invention 1019 or 1020. [The present invention 1031] A method for treating a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of any of the single-chain chimeric polypeptides of the present invention 1001 to 1018 or the composition of the present invention 1019 or 1020. [The present invention 1032] The method of any one of claims 1030 to 1031, wherein said subject has been identified or diagnosed as having cancer, an age-related disease or condition, or an infectious disease. [The present invention 1033] 1032. The method of claim 1032, 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 cancer, lung cancer, renal cell carcinoma, gastic esophageal 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. [The present invention 1034] 1032. The method of claim 1032, 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. [This invention 1035] 1032. The method of claim 1032, 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. [The present invention 1036] A nucleic acid encoding any one of the single-chain chimeric polypeptides of the present invention 1001 to 1018. [This invention 1037] A vector comprising the nucleic acid of the present invention. [The present invention 1038] A cell comprising the nucleic acid of the present invention 1036 or the vector of the present invention 1037. [This invention 1039] 1. A method for producing a single-chain chimeric polypeptide, comprising: culturing the cells of invention 1038 in a culture medium under conditions sufficient to result in the production of said single-chain chimeric polypeptide; recovering said single-chain chimeric polypeptide from said cells and / or said culture medium; A method comprising: [The present invention 1040] A single-chain chimeric polypeptide produced by the method of the present invention. [The present invention 1041] The single-chain chimeric polypeptide of the present invention, wherein the human soluble tissue factor domain does not initiate blood coagulation. [The present invention 1042] 19. The single-chain chimeric polypeptide of any of claims 1001 to 1018, 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] FIG. 1 is a schematic diagram of an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide. [Figure 2] 1 is a chromatograph showing the elution of an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide from an anti-tissue factor antibody affinity column. [Figure 3] 1 is a chromatograph showing the elution of a Superdex 200 Increase 10 / 300 GL gel filtration column loaded with an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide. [Figure 4] Sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) of an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide purified using an anti-tissue factor antibody affinity column. [Figure 5] Figure 1 is a graph showing ELISA quantification of exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptides performed using the method described in Example 1. Purified tissue factor was used as a control. [Figure 6] 1 is a graph showing the ability of exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptides to stimulate CD25 expression in CD4+ T-cells isolated from blood from two donors. The experiment was performed as described in Example 2. [Figure 7] 1 is a graph showing the ability of exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptides to stimulate CD25 expression in CD8+ T-cells isolated from blood from two donors. The experiment was performed as described in Example 2. [Figure 8] 1 is a graph showing the ability of exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptides to stimulate CD69 expression in CD4+ T-cells isolated from blood from two donors. The experiment was performed as described in Example 2. [Figure 9] FIG. 1 is a schematic diagram of an exemplary IL-2 / TF / IL-2 single-chain chimeric polypeptide. [Figure 10] IL-2 activity of IL-2 / TF / IL-2 compared to recombinant IL-2 using a 32D beta cell proliferation assay. [Figure 11] IL-2 activity of IL-2 / TF / IL-2 compared to recombinant IL-2 using a CTLL-2 cell proliferation assay. [Figure 12] Fasting blood glucose levels in ApoE- / - mice fed a standard chow or a high-fat diet and treated with PBS control (untreated) or IL-2 / TF / IL-2 are shown. [Figure 13] Shown are the percentages of CD4+CD25+FoxP3+ T regulatory cells in blood lymphocytes from ApoE- / - mice fed a standard chow or a high-fat diet and treated with PBS control (untreated) or IL-2 / TF / IL-2. [Figure 14] 1 is a line graph showing the chromatographic profile of IL-2 / TF / IL-2 protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody resin. [Figure 15] Analytical SEC profile of IL-2 / TF / IL-2 is shown. [Figure 16A] Figures 16A and 16B show reducing SDS-PAGE analysis of IL-2 / TF / IL-2 before and after deglycosylation. Figure 16A shows reducing SDS-PAGE analysis of IL-2 / TF / IL-2 before deglycosylation. [Figure 16B] FIG. 16B shows a reducing SDS-PAGE analysis of IL-2 / TF / IL-2 after deglycosylation. [Figure 17A] Figures 17A and 17B show the results of immune stimulation in C57BL / 6 mice using IL-2 / TF / IL-2. Figure 17A shows the spleen weight after treatment with IL-2 / TF / IL-2. [Figure 17B] FIG. 17B shows the percentage of immune cell types after IL-2 / TF / IL-2 treatment. [Figure 18] 1 shows upregulation of CD25 expression on CD4+ T cells in mice treated with IL-2 / TF / IL-2. [Figure 19] Pharmacokinetics of IL-2 / TF / IL-2 in C57BL / 6 mice. [Figure 20A] Figures 20A and 20B show the effect of IL-2 / TF / IL-2 in attenuating high-fat-induced atherosclerotic plaque formation in ApoE− / − mice. Figure 20A shows representative images of atherosclerotic plaques from ApoE− / − mice fed a standard chow or a high-fat diet and treated with either PBS control or IL-2 / TF / IL-2. [Figure 20B] FIG. 20B shows the results of quantitative analysis of atherosclerotic plaques in each group. [Figure 21] Fasting blood glucose levels in IL-2 / TF / IL-2 treated mice compared to control treated mice are shown. [Figure 22] The percentage of CD4+CD25+FoxP3+ Tregs in blood lymphocytes from IL-2 / TF / IL-2-treated and control-treated mice is shown. [Figure 23] FIG. 1 is a schematic diagram of an exemplary IL-15 / TF / IL-15 single-chain chimeric polypeptide. [Figure 24] IL-15 activity of IL-15 / TF / IL-15 compared to recombinant IL-15 in a 32D beta cell proliferation assay. [Figure 25]1 is a line graph showing the chromatographic profile of IL-15 / TF / IL-15 protein-containing cell culture supernatant after binding to and elution from an anti-TF antibody resin. [Figure 26A] Figures 26A and 26B show reducing SDS-PAGE analysis of IL-15 / TF / IL-15 before and after deglycosylation. Figure 26A shows reducing SDS-PAGE analysis of IL-15 / TF / IL-15 before deglycosylation. [Figure 26B] FIG. 26B shows a reducing SDS-PAGE analysis of IL-15 / TF / IL-15 after deglycosylation. [Figure 27] 27A-27C are a set of graphs showing immune stimulation in C57BL / 6 mice after treatment with 2t2. [Figure 28] 28A-28C are a set of graphs showing in vivo stimulation of Treg cells, NK cells, and CD8+ T cells in ApoE− / − mice fed a Western diet and treated with 2t2. [Figure 29] Figures 29A and 29B are a pair of graphs showing the induction of splenocyte proliferation by 2t2 in C57BL / 6 mice. [Figure 30] 30A and 30B are a pair of graphs showing in vivo induction of NK cell and CD8+ T cell proliferation in ApoE− / − mice fed a Western diet and treated with 2t2. [Figure 31] 31A-31C are a set of graphs showing amelioration of Western diet-induced hyperglycemia in ApoE− / − mice by 2t2. [Figure 32] Shows upregulation of CD44 memory T cells upon treatment with 2t2. [Figure 33] 1 shows a graph of Factor X (FX) activation after treatment with single-chain or multi-chain chimeric polypeptides. [Figure 34]Figures 34A-34C show human blood lymphocyte pStat5a responses in CD4+CD25hi Treg cells, CD4+CD25- Tcon cells, or CD8+ Tcon cells in response to 2t2 or IL2 treatment. Figure 34A shows pSTAT5 responses in CD4+CD25hi Treg cells. Figure 34B shows pSTAT5 responses in CD4+CD25- Tcon cells. Figure 34C shows pSTAT5 responses in CD8+ Tcon cells. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description Provided herein are single-chain chimeric polypeptides comprising: (i) a first target binding domain (e.g., any of the target binding domains described herein or known in the art), (ii) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art), and (iii) a second target binding domain (e.g., any of the target binding domains described herein or known in the art).

[0035] Also provided herein are compositions comprising any of the single-chain chimeric polypeptides described herein, nucleic acids encoding any of the single-chain chimeric polypeptides described herein, and cells comprising any of the nucleic acids encoding any of the single-chain chimeric polypeptides described herein. Also provided herein are methods of stimulating immune cells and methods of treating a subject in need thereof, comprising the use of any of the single-chain chimeric polypeptides described herein. Also provided herein are methods of producing any of the single-chain chimeric polypeptides described herein.

[0036] In some examples of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide may be from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, from about 50 amino acids to about 700 amino acids, from about 50 amino acids to about 1000 amino acids, About 650 amino acids, about 50 to about 600 amino acids, about 50 to about 550 amino acids, about 50 to about 500 amino acids, about 50 to about 480 amino acids, about 50 to about 460 amino acids, about 50 to about 440 amino acids, about 50 to about 420 amino acids, about 50 to about 400 amino acids, about 50 to about 380 amino acids, about 50 to about 360 amino acids, about 50 to about 340 amino acids, about 50 to about 320 amino acids, about 50 to about 300 amino acids, about 50 to about 280 amino acids, about 50 Amino acids up 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 1 00 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids,About 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 10 00 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 4 ...500 amino acids, about 150 amino acids to about About 420 amino acids, about 150 to about 400 amino acids, about 150 to about 380 amino acids, about 150 to about 360 amino acids, about 150 to about 340 amino acids, about 150 to about 320 amino acids, about 150 to about 300 amino acids, about 150 to about 280 amino acids, about 150 to about 260 amino acids, about 150 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 amino acids amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids,About 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 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 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids,About 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids 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 About 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids,About 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 10 00 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 4 ... About 420 amino acids, about 300 to about 400 amino acids, about 300 to about 380 amino acids, about 300 to about 360 amino acids, about 300 to about 340 amino acids, about 300 to about 320 amino acids, about 320 to about 3000 amino acids, about 320 to about 2500 amino acids, about 320 to about 2000 amino acids, about 320 to about 1500 amino acids, about 320 to about 1000 amino acids, about 320 to about 950 amino acids, about 320 to about 900 amino acids, about 320 to about 850 amino acids, about 3 20 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, of 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 34 0 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 70 0 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids 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 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 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids, about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids,About 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, About 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 1500 amino acids 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 3000 amino acids, about 550 to about 2500 amino acids, about 550 to about 2000 amino acids, about 550 to about 1500 amino acids, about 550 to about 1000 amino acids, about 550 to about 950 amino acids,About 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 30 00 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids,About 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 The single-chain chimeric polypeptides may have a total length of from about 3,000 amino acids, from about 950 amino acids to about 2,500 amino acids, from about 950 amino acids to about 2,000 amino acids, from about 950 amino acids to about 1,500 amino acids, from about 950 amino acids to about 1,000 amino acids, from about 1,000 amino acids to about 3,000 amino acids, from about 1,000 amino acids to about 2,500 amino acids, from about 1,000 amino acids to about 2,000 amino acids, from about 1,000 amino acids to about 1,500 amino acids, from about 1,500 amino acids to about 3,000 amino acids, from about 1,500 amino acids to about 2,500 amino acids, from about 1,500 amino acids to about 2,000 amino acids, from about 2,000 amino acids to about 3,000 amino acids, from about 2,000 amino acids to about 2,500 amino acids, or from about 2,500 amino acids to about 3,000 amino acids. A diagram of an exemplary single-chain chimeric polypeptide provided herein is shown in FIG. 1.

[0037] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are immediately adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments of any of the single-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are immediately adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary target binding domains described herein) and the second target binding domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art).

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

[0039] In some embodiments, the single-chain chimeric polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to (SEQ ID NO: 1).

[0040]

[0041] In some embodiments, the single-chain chimeric polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to (SEQ ID NO: 3).

[0042]

[0043] In some embodiments, the single-chain chimeric polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to (SEQ ID NO: 5).

[0044]

[0045] In some embodiments, the single-chain chimeric polypeptide can comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to (SEQ ID NO: 7).

[0046]

[0047] 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.

[0048] 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.

[0049] The extracellular domain of tissue factor, composed of two fibronectin type III domains, is connected 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, between βC and βD, and between 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 a 17-amino acid β-hairpin between strands β10 and β11, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is absent in the C-terminal domain and is unique to tissue factor. Such a fibronectin type III domain structure is characteristic of the immunoglobulin-like protein family fold and is conserved among a wide variety of extracellular proteins.

[0050] 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 ion transport. 2+It further increases the rate of activation 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.

[0051] 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 in the short linker region between the EGF2 and protease domains. 154 -Arg 152 Activation 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 343It 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.

[0052] Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is important for FVIIa to its cognate substrate. Free FVIIa adopts a stable extended conformation upon membrane binding, 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, demonstrating that tissue factor can fully support FVIIa active site positioning even in the absence of FVIIa-membrane interactions. Additional data showed that tissue factor supported full FVIIa proteolytic activity as long as the tissue factor extracellular domain was somehow tethered to the membrane surface. However, elevating the FVIIa active site beyond 80 Å above the membrane surface significantly reduced the ability of the tissue factor-FVIIa complex to activate FX, but did not reduce tissue factor-FVIIa amidolytic activity.

[0053] 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 well exposed to solvent in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand-binding site spans an extensive region of 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. 133 is 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 to the concave side of the intermodule angle containing Asp. 58 From 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 37The 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:

[0054] 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 the full FVIIa amidolytic activity of small peptidyl substrates but lack the ability to support the 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 that is quite distal to the FVIIa active site. Substitution of the glycine residues with slightly more bulky alanine residues significantly impaired 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.

[0055] In addition, the 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 166are 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 Gla 35 The putative salt bridge formation between tissue factor and the GLA domain of FVIIa would support the notion that tissue factor interaction with the GLA domain of FVIIa regulates substrate recognition. These results suggest that the C-terminal part 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 regulate these interactions either directly or indirectly.

[0056] Soluble tissue factor domain In some embodiments of any of the single-chain chimeric 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. Exemplary soluble human tissue factor domain (SEQ ID NO: 9) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary nucleic acid encoding a soluble human tissue factor domain (SEQ ID NO: 10) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGAC AGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG Exemplary soluble mouse tissue factor domain (SEQ ID NO: 11) agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnap kflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge Exemplary soluble rat tissue factor domain (SEQ ID NO: 12) Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 96) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 97) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0057] In some embodiments, the soluble tissue factor domain may comprise a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 9, 11, 12, 96, or 97. In some embodiments, the soluble tissue factor domain may comprise the sequence of SEQ ID NO: 9, 11, 12, 96, or 97 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.

[0058] 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.

[0059] In some examples of any of the single-chain chimeric polypeptides described herein, the soluble tissue factor domain cannot bind to factor VIIa. In some examples of any of the single-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 single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

[0060] 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.

[0061] 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:96 or SEQ ID NO:97.

[0062] In some examples, the soluble tissue factor domain can be encoded by a nucleic acid comprising a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:10.

[0063] In some embodiments, the soluble tissue factor domain is from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 16 ...85 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 2 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, 0 to about 80 amino acids, about 20 to about 75 amino acids, about 20 to about 70 amino acids, about 20 to about 60 amino acids, about 20 to about 50 amino acids, about 20 to about 40 amino acids, about 20 to about 30 amino acids, about 30 to about 220 amino acids, about 30 to about 215 amino acids, about 30 to about 210 amino acids, about 30 to about 205 amino acids, about 30 to about 200 amino acids, about 30 to about 195 amino acids, about 30 to about 190 amino acids, about 30 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 60 amino acids, about 30 to about 50 amino acids, about 30 to about 40 amino acids, about 40 to about 220 amino acids, about 40 to about 215 amino acids, about 40 to about 210 amino acids, about 40 to about 205 amino acids, About 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about about 220 amino acids, about 50 to about 215 amino acids, about 50 to about 210 amino acids, about 50 to about 205 amino acids, about 50 to about 200 amino acids, about 50 to about 195 amino acids, about 50 to about 190 amino acids, about 50 to about 185 amino acids, about 50 to about 180 amino acids, about 50 to about 175 amino acids, about 50 to about 170 amino acids, about 50 to about 165 amino acids, about 50 to about 160 amino acids, about 50 to about 155 amino acids, about 50 to about 150 amino acids,About 50 to about 145 amino acids, about 50 to about 140 amino acids, about 50 to about 135 amino acids, about 50 to about 130 amino acids, about 50 to about 125 amino acids, about 50 to about 120 amino acids, about 50 to about 115 amino acids, about 50 to about 110 amino acids, about 50 to about 105 amino acids, about 50 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 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 60 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 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 Amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids,About 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 Amino acids, about 70 to about 90 amino acids, about 70 to about 85 amino acids, about 70 to about 80 amino acids, about 80 to about 220 amino acids, about 80 to about 215 amino acids, about 80 to about 210 amino acids, about 80 to about 205 amino acids, about 80 to about 200 amino acids, about 80 to about 195 amino acids, about 80 to about 190 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 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids,About 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 14 ... amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, 10 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids,About 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115, a amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids , about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 1 50 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 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 170 amino acids, about 135 amino acids to about 165 ...65 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 1 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 215 amino acids about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 215 amino acids, 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 20 ... about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 215 amino acids, 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,It may have a total length of about 210 to about 220 amino acids, about 210 to about 215 amino acids, or about 215 to about 220 amino acids.

[0064] In some embodiments, the soluble tissue factor domain may comprise or consist of soluble wild-type human tissue factor (or any sequence therefrom).

[0065] 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 Vol. 27, No. 10, pp. 325-330, 2014, and Priyanka et al., Protein Sci., 2013 Feb; 22(2): 153-167. In some examples, the linker sequence is a synthetic linker sequence.

[0066] Some embodiments of any of the single-chain chimeric polypeptides described herein may 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). Some embodiments of any of the single-chain chimeric polypeptides described herein may 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).

[0067] In some embodiments, the linker sequence may be from 1 to about 100 amino acids, from 1 to about 90 amino acids, from 1 to about 80 amino acids, from 1 to about 70 amino acids, from 1 to about 60 amino acids, from 1 to about 50 amino acids, from 1 to about 45 amino acids, from 1 to about 40 amino acids, from 1 to about 35 amino acids, from 1 to about 30 amino acids, from 1 to about 25 amino acids, from 1 to about 24 amino acids, from 1 to about 22 amino acids, from 1 to about 20 amino acids, from 1 to about 18 amino acids, from 1 to about 16 amino acids, or from 1 to about 2 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 to about 3 About 24 amino acids, about 2 to about 22 amino acids, about 2 to about 20 amino acids, about 2 to about 18 amino acids, about 2 to about 16 amino acids, about 2 to about 14 amino acids, about 2 to about 12 amino acids, about 2 to about 10 amino acids, about 2 to about 8 amino acids, about 2 to about 6 amino acids, about 2 to about 4 amino acids, about 4 to about 100 amino acids, about 4 to about 90 amino acids, about 4 to about 80 amino acids, about 4 to about 70 amino acids, about 4 to about 60 amino acids, about 4 to about 50 amino acids, about 4 amino acids about 4 to about 45 amino acids, about 4 to about 40 amino acids, about 4 to about 35 amino acids, about 4 to about 30 amino acids, about 4 to about 25 amino acids, about 4 to about 24 amino acids, about 4 to about 22 amino acids, about 4 to about 20 amino acids, about 4 to about 18 amino acids, about 4 to about 16 amino acids, about 4 to about 14 amino acids, about 4 to about 12 amino acids, about 4 to about 10 amino acids, about 4 to about 8 amino acids, about 4 to about 6 amino acids, about 6 to about 100 amino acids, about 6 to about 90 amino acids,About 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 1 About 10 amino acids, about 6 to about 8 amino acids, about 8 to about 100 amino acids, about 8 to about 90 amino acids, about 8 to about 80 amino acids, about 8 to about 70 amino acids, about 8 to about 60 amino acids, about 8 to about 50 amino acids, about 8 to about 45 amino acids, about 8 to about 40 amino acids, about 8 to about 35 amino acids, about 8 to about 30 amino acids, about 8 to about 25 amino acids, about 8 to about 24 amino acids, about 8 to about 22 amino acids, about 8 to about 20 amino acids, about 8 to about 18 amino acids , about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 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 to about 45 amino acids, about 12 to about 40 amino acids, about 12 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 5 about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, About 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 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 to about 30 amino acids, about 20 to about 25 amino acids, about 20 to about 24 amino acids, about 20 to about 22 amino acids, about 22 to about 100 amino acids, about 22 to about 90 amino acids, about 22 to about 80 amino acids, about 22 to about 70 amino acids, about 22 to about 60 amino acids, about 22 to about 50 amino acids, about 22 to about 45 amino acids, about 22 to about 40 amino acids, about 22 to about 35 amino acids, about 22 to about 30 amino acids, about 22 to about 25 amino acids, about 22 to about 24 amino acids, about 25 to about 100 amino acids, about 25 to about 90 amino acids, about 25 to about 80 amino acids, about 25 to about 70 amino acids, about 25 to about 60 amino acids, about 25 to about 50 amino acids, about 25 to about 45 amino acids, about 25 to about 40 amino acids, about 25 to about 35 amino acids, about 25 to about 30 amino acids, about 30 to about 100 amino acids, about 30 to about 90 amino acids, about 30 to about 80 amino acids, about 30 to about 70 amino acids, about 30 to about 60 amino acids, 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 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 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids,It may have a total length of 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.

[0068] 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.

[0069] In some embodiments, the linker sequence may comprise or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 13). In some embodiments, the linker sequence may be encoded by a nucleic acid comprising or consisting of GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 14). In some embodiments, the linker sequence may comprise or consist of GGGSGGGS (SEQ ID NO: 15).

[0070] Target Binding Domain In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the additional target binding domain or domains can be an antigen binding domain (e.g., any of the exemplary antigen binding domains described herein or known in the art), a soluble interleukin or cytokine protein (e.g., any of the exemplary soluble interleukin or soluble cytokine proteins described herein), and a soluble interleukin or cytokine receptor (e.g., any of the exemplary soluble interleukin or soluble cytokine receptors described herein).

[0071] In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary 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, CD3ε, and CD3β). 3γ), 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, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHC1 ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, The antibody specifically binds to a target selected from the group consisting of 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.

[0072] In some embodiments of any of the single-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 are each independently from about 5 amino acids to about 1000 amino acids, from about 5 amino acids to about 950 amino acids, from about 5 amino acids to about 900 amino acids, from about 5 amino acids to about 850 amino acids, from about 5 amino acids to about 800 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 650 amino acids, from about 5 amino acids to about 6 ...700 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 650 amino acids, from about 5 amino acids to about 600 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from About 550 amino acids, about 5 to about 500 amino acids, about 5 to about 450 amino acids, about 5 to about 400 amino acids, about 5 to about 350 amino acids, about 5 to about 300 amino acids, about 5 to about 280 amino acids, about 5 to about 260 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 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115 amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about 105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 1 about 95 amino acids, about 5 to about 90 amino acids, about 5 to about 85 amino acids, about 5 to about 80 amino acids, about 5 to about 75 amino acids, about 5 to about 70 amino acids, about 5 to about 65 amino acids, about 5 to about 60 amino acids, about 5 to about 55 amino acids, about 5 to about 50 amino acids, about 5 to about 45 amino acids, about 5 to about 40 amino acids, about 5 to about 35 amino acids, about 5 to about 30 amino acids, about 5 to about 25 amino acids, about 5 to about 20 amino acids, about 5 to about 15 amino acids,About 5 amino acids to about 10 amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids, about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 Amino acids, about 10 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 amino acids to about 950 amino acids, about 15 amino acids to about 900 amino acids, about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700 amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to about 600 amino acids, about 15 amino acids to about 550 amino acids, about 15 amino acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15 amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids, about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino acids, about 15 amino acids to about 260 amino acids, about 15 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids 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 to about 145 amino acids, about 15 to about 140 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 amino acids, about 15 to about 70 amino acids, about 15 to about 65 amino acids, about 15 to about 60 amino acids, about 15 to about 55 amino acids, about 15 to about 50 amino acids, about 15 to about 45 amino acids, about 15 to about 40 amino acids, about 15 to about 35 amino acids, about 15 to about 30 amino acids, about 15 to about 25 amino acids, about 15 to about 20 amino acids, about 20 to about 1000 amino acids, about 20 to about 950 amino acids, about 20 to about 900 amino acids, about 20 to about 850 amino acids,About 20 amino acids to about 800 amino acids, about 20 amino acids to about 750 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids, about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 1000 amino acids, about 25 amino acids to about 950 amino acids, about 25 amino acids to about 900 amino acids, about 25 amino acids to about 850 amino acids, about 25 amino acids to about 800 amino acids, about 25 amino acids to about 750 amino acids, about 25 amino acids to about 700 amino acids, about 25 amino acids to about 650 amino acids,About 25 amino acids to about 600 amino acids, about 25 amino acids to about 550 amino acids, about 25 amino acids to about 500 amino acids, about 25 amino acids to about 450 amino acids, about 25 amino acids to about 400 amino acids, about 25 amino acids to about 350 amino acids, about 25 amino acids to about 300 amino acids, about 25 amino acids to about 280 amino acids, about 25 amino acids to about 260 amino acids, about 25 amino acids to about 240 amino acids, about 25 amino acids to about 220 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino acids to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25 amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 2 5 amino acids to about 110 amino acids, about 25 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 5 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 1000 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 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, M 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 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 65 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 55 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, 35 amino acids to about 1000 amino acids, about 35 amino acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35 amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids, about 35 amino acids to about 750 amino acids, about 35 amino acids to about 700 amino acids, about 35 amino acids to about 650 amino acids, about 35 amino acids to about 600 amino acids, about 35 amino acids to about 550 amino acids, about 35 amino acids to about 500 amino acids, about 35 amino acids to about 450 amino acids, about 35 amino acids to about 400 amino acids, about 35 amino acids to about 350 amino acids, about 35 amino acids to about 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 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, 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 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40 amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids,About 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 Amino acids up to about 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45 amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids, about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino acids, about 45 amino acids to about 30 0 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 up to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids, about 45 amino acids to about 120 amino acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids,About 45 to about 75 amino acids, about 45 to about 70 amino acids, about 45 to about 65 amino acids, about 45 to about 60 amino acids, about 45 to about 55 amino acids, about 45 to about 50 amino acids, about 50 to about 1000 amino acids, about 50 to about 950 amino acids, about 50 to about 900 amino acids, about 50 to about 850 amino acids, about 50 to about 800 amino acids, about 50 to about 750 amino acids, about 50 to about 700 amino acids, about 50 to about 650 amino acids, about 50 to about 600 amino acids amino acids, about 50 to about 550 amino acids, about 50 to about 500 amino acids, about 50 to about 450 amino acids, about 50 to about 400 amino acids, about 50 to about 350 amino acids, about 50 to about 300 amino acids, about 50 to about 280 amino acids, about 50 to about 260 amino acids, about 50 to about 240 amino acids, about 50 to about 220 amino acids, about 50 to about 200 amino acids, about 50 to about 195 amino acids, about 50 to about 190 amino acids, about 50 to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids , about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids,About 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids, about 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 22 0 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids to about 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 350 amino acids, about 60 amino acids to about 300 amino acids,About 60 amino acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60 amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, of 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, about 60 to about 110 amino acids, about 60 to about 105 amino acids, about 60 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 6 50 amino acids, about 65 amino acids to about 600 amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to about 500 amino acids, about 65 amino acids to about 450 amino acids, about 65 amino acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65 amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids, about 65 amino acids to about 260 amino acids, about 65 amino acids to about 240 amino acids, about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 19 ... about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 135 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids,About 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 950 amino acids, about 70 amino acids to about 900 amino acids, about 70 amino acids to about 850 amino acids, about 70 amino acids to about 800 amino acids, about 70 amino acids to about 750 amino acids amino acids, about 70 to about 700 amino acids, about 70 to about 650 amino acids, about 70 to about 600 amino acids, about 70 to about 550 amino acids, about 70 to about 500 amino acids, about 70 to about 450 amino acids, about 70 to about 400 amino acids, about 70 to about 350 amino acids, about 70 to about 300 amino acids, about 70 to about 280 amino acids, about 70 to about 260 amino acids, about 70 to about 240 amino acids, about 70 to about 220 amino acids, about 70 to about 200 amino acids, about 70 to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 7 0 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 1000 amino acids, about 75 amino acids to about 950 amino acids, about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino acids, about 75 amino acids to about 800 amino acids,About 75 amino acids to about 750 amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75 amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids to about 350 amino acids, about 75 amino acids to about 300 amino acids, about 75 amino acids to about 280 amino acids, about 75 amino acids to about 260 amino acids, about 75 amino acids to about 240 amino acids, about 75 amino acids to about 220 amino acids, about 75 amino acids to about 200 Amino acids, about 75 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, about 75 to about 145 amino acids, about 75 to about 140 amino acids, about 75 to about 135 amino acids, about 75 to about 130 amino acids, about 75 amino acids about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 1000 amino acids, about 80 amino acids to about 950 amino acids, about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino acids, about 80 amino acids to about 800 amino acids, about 80 amino acids to about 750 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids, about 80 amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids, about 80 amino acids to about 350 amino acids, about 80 amino acids to about 300 amino acids, about 80 amino acids to about 280 amino acids, about 80 amino acids to about 260 amino acids, about 80 amino acids to about 240 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 200 amino acids,About 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 Amino acids, about 80 to about 120 amino acids, about 80 to about 115 amino acids, about 80 to about 110 amino acids, about 80 to about 105 amino acids, about 80 to about 100 amino acids, about 80 to about 95 amino acids, about 80 to about 90 amino acids, about 80 to about 85 amino acids, about 85 to about 1000 amino acids, about 85 to about 950 amino acids, about 85 to about 900 amino acids, about 85 to about 850 amino acids, about 85 to about 800 amino acids, about 85 to about 750 amino acids, about 85 to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 260 amino acids, about 85 amino acids to about 240 amino acids, about 85 amino acids to about 220 amino acids, about 85 amino acids to about 200 amino acids, about 85 amino acids to about 195 ... amino acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85 amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155 amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to about 145 amino acids, about 85 amino acids to about 140 amino acids, about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85 amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids,About 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90 amino acids to about 850 amino acids, about 90 amino acids to about 800 amino acids, about 90 amino acids to about 750 amino acids, about 90 amino acids to about 700 amino acids, about 90 amino acids to about 650 amino acids, about 90 amino acids to about 600 amino acids, about 90 amino acids to about 550 amino acids, about 90 amino acids to about 500 amino acids, about 90 amino acids to about 450 amino acids, about 90 amino acids to about 400 amino acids, about 90 amino acids to about 350 amino acids, about 90 amino acids to about 300 amino acids, about 90 amino acids to about 280 amino acids, about 90 amino acids to about 260 amino acids, about 90 amino acids to about 240 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, About 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 ...25 amino acids, about 90 amino acids to about 120 amino acids, about 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, of 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, 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids, about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 Amino acids up to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 1 00 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 18 ... About 175 amino acids, about 105 amino acids to about 170 amino acids, about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130 amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids amino acids to about 1000 amino acids, about 110 amino acids to about 950 amino acids, about 110 amino acids to about 900 amino acids, about 110 amino acids to about 850 amino acids, about 110 amino acids to about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110 amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids, about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550 amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to about 450 amino acids, about 110 amino acids to about 400 amino acids, about 110 amino acids to about 350 amino acids,About 110 amino acids to about 300 amino acids, about 110 amino acids to about 280 amino acids, about 110 amino acids to about 260 amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 Amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115 amino acids 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 up to about 220 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids,About 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids, about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to about 600 amino acids, about 120 amino acids to about 550 Amino acids, about 120 amino acids to about 500 amino acids, about 120 amino acids to about 450 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350 amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids to about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120 amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 ... about 180 amino acids, about 120 amino acids to about 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 1000 amino acids, about 125 amino acids to about 950 amino acids, about 125 Amino acids up to about 900 amino acids, about 125 amino acids to about 850 amino acids, about 125 amino acids to about 800 amino acids, about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700 amino acids, about 125 amino acids to about 650 amino acids, about 125 amino acids to about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125 amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350 amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to about 280 amino acids,About 125 amino acids to about 260 amino acids, about 125 amino acids to about 240 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids 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 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 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids, about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900 amino acids, about 135 amino acids to about 850 amino acids,About 135 amino acids to about 800 amino acids, about 135 amino acids to about 750 amino acids, about 135 amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids, about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550 amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to about 450 amino acids, about 135 amino acids or more, 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 850 amino acids amino acids to about 600 amino acids, about 140 amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 350 amino acids, about 140 amino acids to about 300 amino acids, about 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 19 ... 0 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids, about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900 amino acids, about 145 amino acids to about 850 amino acids, about 145 amino acids to about 800 amino acids,About 145 amino acids to about 750 amino acids, about 145 amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids, about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550 amino acids, about 145 amino acids to about 500 amino acids, about 145 amino acids to about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145 amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids, about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260 amino acids, about 145 amino acids to about 240 amino acids, about 145 amino acids to about 220 amino acids 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 1000 amino acids 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 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 1000 amino acids,About 155 amino acids to about 950 amino acids, about 155 amino acids to about 900 amino acids, about 155 amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids, about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700 amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155 amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids, about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350 amino acids, about 155 amino acids to about 300 amino acids amino acids, about 155 amino acids to about 280 amino acids, about 155 amino acids to about 260 amino acids, about 155 amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to about 950 amino acids, about 160 amino acids to about 900 amino acids, about 160 amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids, about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700 amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids, about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350 amino acids, about 160 amino acids amino acids to about 300 amino acids, about 160 amino acids to about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 1000 amino acids,About 165 amino acids to about 950 amino acids, about 165 amino acids to about 900 amino acids, about 165 amino acids to about 850 amino acids, about 165 amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids, about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650 amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165 amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids, about 165 amino acids to about 350 amino acids, about 165 amino acids to about 300 amino acids 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 1000 amino acids 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 amino acids to about 260 amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175 amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids, about 175 amino acids to about 800 amino acids,About 175 amino acids to about 750 amino acids, about 175 amino acids to about 700 amino acids, about 175 amino acids to about 650 amino acids, about 175 amino acids to about 600 amino acids, about 175 amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids, about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175 amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids, about 175 amino acids to about 220 amino acids 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 1000 amino acids 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 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, ~ About 190 amino acids, about 190 to about 1000 amino acids, about 190 to about 950 amino acids, about 190 to about 900 amino acids, about 190 to about 850 amino acids, about 190 to about 800 amino acids, about 190 to about 750 amino acids, about 190 to about 700 amino acids, about 190 to about 650 amino acids, about 190 to about 600 amino acids, about 190 to about 550 amino acids, about 190 to about 500 amino acids, about 190 to about 450 amino acids, about 190 to about 400 amino acids, about 1 90 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 2 ... 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,The total number of amino acids may be 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.

[0073] Any of the target binding domains described herein may be used in combination with 1×10 -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 about 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.

[0074] 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, from about 1 pM to about 850 pM, from about 1 pM to about 800 pM, from about 1 pM to about 750 pM, from about 1 pM to about 700 pM, from about 1 pM to about 650 pM, from about 1 pM to about 600 pM, from about 1 pM to about 550 pM, from about 1 pM to about 500 pM, from about 1 pM to about 450 pM, from about 1 pM to about 5 ...500 pM, from about 1 pM M ~ about 400pM, about 1pM - about 350pM, about 1pM - about 300pM, about 1pM - about 250pM, about 1pM - about 200pM, about 1pM - Approximately 150pM, approximately 1pM to approximately 100pM, approximately 1pM to approximately 90pM, approximately 1pM to approximately 80pM, approximately 1pM to approximately 70pM, approximately 1pM to approximately 60pM , about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5pM, about 1pM to about 4pM, about 1pM to about 3pM, about 1pM to about 2pM, about 2pM to about 30nM, about 2pM to about 25nM, about 2pM to about 2 0nM, about 2pM to about 15nM, about 2pM to about 10nM, about 2pM to about 5nM, about 2pM to about 2nM, about 2pM to about 1nM, about 2pM to about 950pM, about 2pM to about 900pM, about 2pM to about 850pM, about 2pM to about 800pM, about 2pM to about 750pM, about 2pM to about 70 0pM, about 2pM to about 650pM, about 2pM to about 600pM, about 2pM to about 550pM, about 2pM to about 500pM, about 2pM to about 450p M, about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2pM to about 150pM, about 2pM to about 100pM, about 2pM to about 90pM, about 2pM to about 80pM, about 2pM to about 70pM, about 2pM to about 60pM, about 2pM to about 50pM, about 2pM to about 40pM, about 2pM to about 30pM, about 2pM to about 20pM, about 2pM to about 10pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5pM to about 15nM, about 5pM to about 10nM, about 5pM to about 5nM, about 5pM to about 2nM, about 5pM to about 1nM, about 5pM to about 950pM,Approximately 5pM to approximately 900pM, approximately 5pM to approximately 850pM, approximately 5pM to approximately 800pM, 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~ Approximately 400pM, approximately 5pM to approximately 350pM, approximately 5pM to approximately 300pM, approximately 5pM to approximately 250pM, approximately 5pM to approximately 200pM, approximately 5pM to approximately 150pM, approximately 5pM to approximately 100pM, approximately 5pM to approximately 90pM, approximately 5pM to approximately 80pM, approximately 5pM to approximately 70pM, approximately 5pM to approximately 60pM, approximately 5 pM ~ approximately 50 pM, approximately 5 pM ~ approximately 40 pM, approximately 5 pM ~ approximately 30 pM, approximately 5 pM ~ approximately 20 pM, approximately 5 pM ~ approximately 10 pM, approximately 10 pM ~ approximately 30 nM, approximately 10 pM ~ approximately 25 nM, approximately 10 pM ~ approximately 20 nM, approximately 10 pM ~ approximately 15 nM, approximately 10 pM ~ approximately 10 nM, approximately 10 pM ~ approximately 5 nM, approximately 10pM to approximately 2nm, approximately 10pM to approximately 1nm, approximately 10pM to approximately 950pM, approximately 10pM to approximately 900pM, approximately 10pM to approximately 850pM, approximately 10pM to approximately 800pM, approximately 10pM to approximately 750pM, approximately 10pM to approximately 700pM, approximately 10pM to approximately 650pM, approximately 10pM to approximately 600pM Approximately 10 pM to approximately 550 pM, approximately 10 pM to approximately 500 pM, approximately 10 pM to approximately 450 pM, approximately 10 pM to approximately 400 pM, approximately 10 pM to approximately 350 pM, approximately 10 pM to approximately 300 pM, approximately 10 pM to approximately 250 pM, approximately 10 pM to approximately 200 pM, approximately 10 pM to approximately 150 pM, approximately 10 pM to approximately 1 0pM, approximately 10pM to approximately 90pM, approximately 10pM to approximately 80pM, approximately 10pM 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 15 pM to approximately 20 nm, approximately 15 pM to approximately 15 nm, approximately 15 pM 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 p M~approx. 750pM, approx. 15pM~approx. 700pM, approx. 15pM~approx. 650pM, approx. 15pM~approx. 600pM, approx. 15pM~approx. 550pM, approx. 15pM~approx. 500pM, approx. 15pM~approx. 450pM, approx. 15pM~approx. 400pM, approx. 15pM~approx. 350pM, approx. 15pM~approx. 300pMApproximately 15pM to approximately 250pM, approximately 15pM to approximately 200pM, approximately 15pM to approximately 150pM, approximately 15pM to approximately 100pM, approximately 15pM to approximately 90pM, approximately 15pM to approximately 80pM, approximately 15pM to approximately 70pM, approximately 15pM to approximately 60pM, approximately 15pM to approximately 50pM, approximately 15pM to approximately 40pM. Approximately 15 pM to approximately 30 pM, approximately 15 pM to approximately 20 pM, approximately 20 pM to approximately 30 nmM, approximately 20 pM to approximately 25 nmM, approximately 20 pM to approximately 20 nmM, approximately 20 pM to approximately 15 nmM, approximately 20 pM to approximately 10 nmM, approximately 20 pM to approximately 5 nmM, approximately 20 pM to approximately 2 nmM, approximately 20 pM to approximately 1 nmM, approximately 20 pM to approximately 950pM, approximately 20pM to approximately 900pM, approximately 20pM to approximately 850pM, approximately 20pM to approximately 800pM, approximately 20pM to approximately 750pM, approximately 20pM to approximately 700pM, approximately 20pM to approximately 650pM, approximately 20pM to approximately 600pM, approximately 20pM to approximately 550pM, approximately 20pM to approximately 500pM, approximately 20pM to approximately 450pM, approximately 20pM to approximately 400pM, approximately 20pM to approximately 350pM, 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 M, approximately 20pM to approximately 70pM, approximately 20pM to approximately 60pM, approximately 20pM to approximately 50pM, approximately 20pM to approximately 40pM, approximately 20pM to approximately 30pM, approximately 30pM to approximately 30nM, approximately 30pM to approximately 25nM, approximately 30pM to approximately 30nM, approximately 30pM to approximately 15nM, approximately 30pM to approximately 10nM, approximately 3 0pM to approximately 5nm, approximately 30pM to approximately 2nm, approximately 30pM to approximately 1nm, 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 3 0pM~approx. 600pM, approx. 30pM~approx. 550pM, approx. 30pM~approx. 500pM, approx. 30pM~approx. 450pM, approx. 30pM~approx. 400pM, approx. 30pM~approx. 350pM, approx. 30pM~approx. 300pM, approx. 30pM~approx. 250pM, approx. 30pM~approx. 200pM, approx. 30pM~approx. 150pM 0pM, approximately 30pM to approximately 100pM, approximately 30pM to approximately 90pM, approximately 30pM to approximately 80pM, approximately 30pM to approximately 70pM, approximately 30pM to approximately 60pM, approximately 30pM to approximately 50pM, approximately 30pM to approximately 40pM, approximately 40pM to approximately 30nm, approximately 40pM to approximately 25nm, approximately 40pM to approximately 30nm.Approximately 40 pM to approximately 15 nm, approximately 40 pM to approximately 10 nm, approximately 40 pM to 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 40pM to approximately 700pM, approximately 40pM to approximately 650pM, approximately 40pM to 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 M, approximately 40pM to approximately 200pM, approximately 40pM to approximately 150pM, approximately 40pM to approximately 100pM, approximately 40pM to approximately 90pM, approximately 40pM to approximately 80pM, approximately 40pM to approximately 70pM, approximately 40pM to approximately 60pM, approximately 40pM to approximately 50pM, approximately 50pM to approximately 30nm, approximately 50pM to approximately 25nm. Approximately 50 pM to approximately 30 nm, approximately 50 pM to approximately 15 nm, approximately 50 pM to approximately 10 nm, approximately 50 pM to approximately 5 nm, approximately 50 pM to approximately 2 nm, approximately 50 pM to approximately 1 nm, approximately 50 pM to approximately 950 pM, approximately 50 pM to approximately 900 pM, approximately 50 pM to approximately 850 pM, approximately 50 pM to approximately 800 pM, approximately 50 p M~approx. 750pM, approx. 50pM~approx. 700pM, approx. 50pM~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 Approximately 50 pM to approximately 250 pM, approximately 50 pM to approximately 200 pM, approximately 50 pM to approximately 150 pM, approximately 50 pM to approximately 100 pM, approximately 50 pM to approximately 90 pM, approximately 50 pM to approximately 80 pM, approximately 50 pM to approximately 70 pM, approximately 50 pM to approximately 60 pM, approximately 60 pM to approximately 30 nM, approximately 60 pM to approximately 25 nM. Approximately 60 pM to approximately 30 nm, approximately 60 pM to approximately 15 nm, approximately 60 pM to approximately 10 nm, approximately 60 pM to approximately 5 nm, approximately 60 pM to approximately 2 nm, approximately 60 pM to approximately 1 nm, approximately 60 pM to approximately 950 pM, approximately 60 pM to approximately 900 pM, approximately 60 pM to approximately 850 pM, approximately 60 pM to approximately 800 pM, approximately 60 p M~approx. 750pM, approx. 60pM~approx. 700pM, approx. 60pM~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. 300pMApproximately 60 pM to approximately 250 pM, approximately 60 pM to approximately 200 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 70pM ~ approximately 15nM, approximately 70pM ~ approximately 10nM, approximately 70pM ~ approximately 5nM, approximately 70pM ~ approximately 2nM, approximately 70pM ~ approximately 1nM, approximately 70pM ~ approximately 950pM, approximately 70pM ~ approximately 900pM, approximately 70pM ~ approximately 850pM, approximately 70pM ~ approximately 800pM, approximately 70pM ~ approximately 750pM, approximately 70pM M~approx. 700pM, 70pM~approx. 650pM, 70pM~approx. 600pM, 70pM~approx. 550pM, 70pM~approx. 500pM, 70pM~approx. 450pM, 70pM~approx. 400pM, 70pM~approx. 350pM, 70pM~approx. 300pM, 70pM~approx. 250pM Approximately 70 pM to approximately 200 pM, approximately 70 pM to approximately 150 pM, approximately 70 pM to approximately 100 pM, approximately 70 pM to approximately 90 pM, approximately 70 pM to approximately 80 pM, approximately 80 pM to approximately 30 nM, approximately 80 pM to approximately 25 nM, approximately 80 pM to approximately 30 nM, approximately 80 pM to approximately 15 nM, approximately 80 pM to approximately 10 nM, approximately 80pM ~ approximately 5nm, approximately 80pM ~ approximately 2nm, approximately 80pM ~ approximately 1nm, approximately 80pM ~ approximately 950pM, approximately 80pM ~ approximately 900pM, approximately 80pM ~ approximately 850pM, approximately 80pM ~ approximately 800pM, approximately 80pM ~ approximately 750pM, approximately 80pM ~ approximately 700pM, approximately 80pM ~ approximately 650pM, approximately 8 0pM~approx. 600pM, approx. 80pM~approx. 550pM, approx. 80pM~approx. 500pM, approx. 80pM~approx. 450pM, approx. 80pM~approx. 400pM, approx. 80pM~approx. 350pM, approx. 80pM~approx. 300pM, approx. 80pM~approx. 250pM, approx. 80pM~approx. 200pM, approx. 80pM~approx. 150pM pM, 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 M~approx. 950pM, approx. 90pM~approx. 900pM, approx. 90pM~approx. 850pM, approx. 90pM~approx. 800pM, approx. 90pM~approx. 750pM, approx. 90pM~approx. 700pM, approx. 90pM~approx. 650pM, approx. 90pM~approx. 600pM, approx. 90pM~approx. 550pM, approx. 90pM~approx. 500pMApproximately 90 pM to approximately 450 pM, approximately 90 pM to approximately 400 pM, approximately 90 pM to approximately 350 pM, approximately 90 pM to approximately 300 pM, approximately 90 pM to approximately 250 pM, approximately 90 pM to approximately 200 pM, approximately 90 pM to approximately 150 pM, approximately 90 pM to approximately 100 pM, approximately 100 pM to approximately 30 nM, approximately 100 pM to approximately 2... 5 nM, approximately 100pM to approximately 30nM, approximately 100pM to approximately 15nM, approximately 100pM to approximately 10nM, approximately 100pM to approximately 5nM, approximately 100pM to approximately 2nM, approximately 100pM to approximately 1nM, approximately 100pM to approximately 950pM, approximately 100pM to approximately 900pM, approximately 100pM to approximately 850pM, approximately 10 0pM~approx. 800pM, approx. 100pM~approx. 750pM, approx. 100pM~approx. 700pM, approx. 100pM~approx. 650pM, approx. 100pM~approx. 600pM, approx. 100pM~approx. 550pM, approx. 100pM~approx. 500pM, approx. 100pM~approx. 450pM, approx. 100pM~approx. 400pM, approx. 100pM~approx. 350pM, approx. 100pM~approx. 300pM, approx. 100pM~approx. 250pM, approx. 100pM~approx. 200pM, approx. 100pM~approx. 150pM, approx. 150pM~approx. 300pM, approx. 150pM~approx. 150pM, approx. 150pM~approx. 150pM, approx. 150pM~approx. 150pM 0pM ~ approximately 10nM, approximately 150pM ~ approximately 5nM, approximately 150pM ~ approximately 2nM, approximately 150pM ~ approximately 1nM, approximately 150pM ~ approximately 950pM, approximately 150pM ~ approximately 900pM, approximately 150pM ~ approximately 850pM, approximately 150pM ~ approximately 800pM, approximately 150pM ~ approximately 750pM, approximately 150pM ~ approximately 700pM, approximately 150pM to approximately 650pM, approximately 150pM to approximately 600pM, approximately 150pM to approximately 550pM, approximately 150pM to approximately 500pM, approximately 150pM to approximately 450pM, approximately 150pM to approximately 400pM, approximately 150pM to approximately 350pM, approximately 150pM to approximately 300pM, approximately 150pM ~approximately 250 pM, approximately 150 pM ~ approximately 200 pM, approximately 200 pM ~ approximately 30 nm, approximately 200 pM ~ approximately 25 nm, approximately 200 pM ~ approximately 30 nm, approximately 200 pM ~ approximately 15 nm, approximately 200 pM ~ approximately 10 nm, approximately 200 pM ~ approximately 5 nm, approximately 200 pM ~ approximately 2 nm, approximately 200 pM ~ approximately 1 nm, 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, approximately 200pM to approximately 550pM Approximately 200pM to 500pM, approximately 200pM to 450pM, approximately 200pM to 400pM, approximately 200pM to 350pM, approximately 200pM to 300pM, approximately 200pM to 250pM, approximately 300pM to 30nm, approximately 300pM to 25nm, approximately 300pM to 30nm.Approximately 300pM to approximately 15nm, approximately 300pM to approximately 10nm, approximately 300pM to approximately 5nm, approximately 300pM to approximately 2nm, approximately 300pM to approximately 1nm, approximately 300pM to approximately 950pM, approximately 300pM to approximately 900pM, approximately 300pM to approximately 850pM, approximately 300pM to approximately 800pM, approximately 300pM~ 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 to approximately 350pM, approximately 400pM ~approx. 30 nm, approx. 400 pM ~ approx. 25 nm, approx. 400 pM ~ approx. 30 nm, approx. 400 pM ~ approx. 15 nm, approx. 400 pM ~ approx. 10 nm, approx. 400 pM ~ approx. 5 nm, approx. 400 pM ~ approx. 2 nm, approx. 400 pM ~ approx. 1 nm, approx. 400 pM ~ approx. 950 pM, approx. 400 pM ~ approx. 900 pM, approx. 4 00pM~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. 500pM~approx. 30nm, approx. 5 00pM ~ 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, approximately 500pM ~ approximately 850pM pM, 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 to approximately 800pM, approximately 600pM ~750pM, approximately 600pM~approx. 700pM, approximately 600pM~approx. 650pM, approximately 700pM~approx. 30nM, approximately 700pM~approx. 25nM, approximately 700pM~approx. 30nM, approximately 700pM~approx. 15nM, approximately 700pM~approx. 10nM, approximately 700pM~approx. 5nM, approximately 700pM~approx. 2nMApproximately 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 2 nm, approximately 800 pM M ~ approximately 1 nmM, approximately 800 pM ~ approximately 950 pM, approximately 800 pM ~ approximately 900 pM, approximately 800 pM ~ approximately 850 pM, approximately 900 pM ~ approximately 30 nmM, approximately 900 pM ~ approximately 25 nmM, approximately 900 pM ~ approximately 30 nmM, approximately 900 pM ~ approximately 15 nmM, approximately 900 pM ~ approximately 10 nmM, approximately 900 pM ~ approximately 5 nmM, approximately 900 pM ~ approximately 2 nmM, approximately 900 pM ~ approximately 1 nmM, approximately 900 pM ~ approximately 950 pM, approximately 1 nmM ~ approximately 30 nmM, approximately 1 nM ~ approximately 25 nM, approximately 1 nM ~ approximately 20 nM, approximately 1 nM ~ approximately 15 nM, approximately 1 nM ~ approximately 10 nM, approximately 1 nM ~ approximately 5 nM, approximately 2 nM ~ approximately 30 nM, approximately 2 nM ~ approximately 25 nM, approximately 2 nM ~ approximately 20 nM, approximately 2 nM ~ approximately 15 nM, approximately 2 nM ~ approximately 10 nM, approximately 2 nM ~ approximately 5 nM, approximately 4 nM ~ approximately 30 nM, approximately 4 nM ~ approximately 25 nM, approximately 4 nM ~ approximately 20 nM, approximately 4 nM ~ approximately 15 nM, approximately 4 nM ~ approximately 10 nM, approximately 4 n M~approx. 5nM, approx. 5nM~approx. 30nM, approx. 5nM~approx. 25nM, approx. 5nM~approx. 20nM, approx. 5nM~approx. 15nM, approx. 5nM~approx. 10nM, approx. 10nM~approx. 30nM, approx. 15nM~approx. 25nM, approx. 15nM~approx. 20nM, approx. 20nM~approx. 30nM, approx. 20nM~approx. 30nM, approx. 20nM~approx. 25nM) K, D The combination of the でその standard and the することができる.

[0075] 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, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about ~about 6nM, about 3nM to about 5nM, about 3nM to about 4nM, about 4nM to about 10nM, about 4nM to about 9nM, about 4nM to about 8nM, about 4nM ~about 7nM, about 4nM to about 6nM, about 4nM to about 5nM, about 5nM to about 10nM, about 5nM to about 9nM, about 5nM to about 8nM, about 5nM Approximately 7nM, approximately 5nM to approximately 6nM, approximately 6nM to approximately 10nM, approximately 6nM to approximately 9nM, approximately 6nM to approximately 8nM, approximately 6nM to approximately 7nM, approximately 7nM to approximately K of 10nM, about 7nM to about 9nM, about 7nM to about 8nM, about 8nM to about 10nM, about 8nM to about 9nM, and about 9nM to about 10nM) D can bind to its target.

[0076] 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.).

[0077] antigen-binding domain In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of these single-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these single-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0078] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0079] In some embodiments of any of the single-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 single-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each antigen-binding domains.

[0080] In some embodiments of any of the single-chain chimeric polypeptides described herein, the antigen-binding domain is an scFv or single-domain antibody (e.g., V H H or V NAR domain).

[0081] 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, for example, those described in U.S. Patent Application Publication No. 2016 / 0340413), PDL-1 (see, for example, those described in Drees Patent Application Publication No. 2016 / 0340413), CD123 (see, for example, those described in WO2014 / 130635), IL-1R (see, for example, those described in U.S. Patent No. 8,741,604), IL-1 (see, for example, those described in WO2014 / 095808), VEGF (see, for example, those described in U.S. Patent No. 9,090,684), IL-6R (see, for example, those described in U.S. Patent No. 7,482,436), IL-4 (see, for example, those described in U.S. Patent Application Publication No. 2012 / 0171197), IL-10 (see, for example, those described in U.S. Patent Application Publication No. 2016 / 0340413), PDL-1 (see, for example, those described in Drees Patent Application Publication No. 2016 / 0340413), et al., Protein Express. Purif. 94:60-66, 2014), TIGIT (see, e.g., as described in U.S. Patent Application Publication No. 2017 / 0198042), PD-1 (see, e.g., as described in U.S. Patent No. 7,488,802), TIM3 (see, e.g., as described in U.S. Patent No. 8,552,156), CTLA4 (see, e.g., as described in WO2012 / 120125), MICA (see, e.g., as described in WO2016 / 154585), MICB (see, e.g., as described in U.S. Patent No. 8,753,640), IL-6 (see, e.g., as described in Gejima et al., Human Antibodies 11(4):121-129, 2002), IL-8 (see, e.g., as described in U.S. Patent No. 6,117,980), TNFα (see, e.g., as described in Geng et al., al., Immunol. Res.62(3):377-385, 2015), CD26a (see, for example, those described in WO2017 / 189526), ​​CD36 (see, for example, those described in U.S. Patent Application Publication No. 2015 / 0259429), ULBP2 (see, for example, those described in U.S. Patent No. 9,273,136), CD30 (see, for example, Homach et al. 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, e.g., as described in U.S. Patent Application Publication No. 2013 / 0274446), HER2 (see, e.g., as described in Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3 (see, e.g., as described in U.S. Patent No. 9,505,843), PSMA (see, e.g., as described in Parker et al., Protein Expr. Purif. 89(2):136-145, 2013), CEA (see, e.g., as described in WO1995 / 015341), B7H3 (see, e.g., as described in U.S. Patent No. 9,371,395), EPCAM (see, e.g., as described in WO2014 / 159531), BCMA (see, e.g., as described in Smith et al., al.,Mol.Ther.26(6):1447-1456,2018), P-cadherin (see, for example, those described in U.S. Pat. No. 7,452,537), CEACAM5 (see, for example, those described in U.S. Pat. No. 9,617,345), UL16 binding protein (see, for example, those described in WO2017 / 083612), HLA-DR (see, for example, Pistillo et al. 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 PDGFDD (see, e.g., those described in U.S. Patent No. 9,441,034).

[0082] The antigen binding domains present in any of the single-chain chimeric polypeptides described herein are each independently selected from the group consisting of a VHH domain, a VNAR domain, and an scFv.

[0083] In some embodiments, one or more of the first target binding domain, the second target binding domain, and / or the one or more additional antigen binding domains can be an anti-CD3 scFv. In some embodiments, the anti-CD3 scFv comprises a heavy chain variable domain comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 16). and / or a light chain variable domain comprising a sequence 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 QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 17). In some embodiments, an scFv (e.g., any of the scFvs described herein) may comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the heavy chain variable domain and the light chain variable domain.In some embodiments, anti-CD3 The scFv can be synthesized by a nucleic acid comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACTACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC (SEQ ID NO: 18). The nucleic acid sequence may comprise a heavy chain variable domain encoded by a nucleic acid sequence 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 CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGT (SEQ ID NO: 19).

[0084] In some embodiments, the anti-CD3 scFv may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGGGGSGGGGSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (SEQ ID NO: 20). In some embodiments, the anti-CD3 scFv may comprise the six CDRs present in SEQ ID NO: 20.

[0085] In some embodiments, the anti-CD3 scFv may comprise a sequence encoded by a nucleic acid 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 (SEQ ID NO: 21).

[0086] In some embodiments, one or more of the first target binding domain, the second target binding domain, and / or the one or more additional antigen binding domains can be an anti-CD28 scFv. In some embodiments, the anti-CD28 scFv comprises a heavy chain variable domain comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (SEQ ID NO: 22); and / or can comprise a light chain variable domain comprising a sequence 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 VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSS (SEQ ID NO: 23).In some embodiments, the anti-CD28 scFv comprises a heavy chain variable domain encoded by a nucleic acid comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTCTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCCTAAACTGTGTATCTACTCCACAAGCAATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGG (SEQ ID NO: 24), and / or CTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACCAAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGGAACTACTTTAACAGTGAGCTCC (SEQ ID NO: 25).

[0087] In some embodiments, the anti-CD28 scFv may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSSGGGGSGGGGSGGGGSDIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (SEQ ID NO:26). In some embodiments, the anti-CD28 scFv may comprise the six CDRs present in SEQ ID NO:26.

[0088] In some embodiments, the anti-CD28 scFv may comprise a sequence encoded by a nucleic acid 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 (SEQ ID NO: 27).

[0089] In some embodiments, any of the antigen-binding domains described herein is a BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. Additional examples of antigen-binding domains that can be used in any of the single-chain chimeric polypeptides are known in the art.

[0090] VHH domains are single monomeric variable antibody domains that can be found in camelids. NAR VHH domains are single monomeric variable antibody domains that can be found in cartilaginous fish. NARNon-limiting aspects of the domain 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.

[0091] In some embodiments, both of the antigen-binding domains in the single-chain chimeric polypeptides described herein are VHH domains, or at least one antigen-binding domain is a VHH domain. In some embodiments, both of the antigen-binding domains in the single-chain chimeric polypeptides described herein are VNAR domains, or at least one antigen-binding domain is a VNAR domain. In some embodiments, both of the antigen-binding domains in the single-chain chimeric polypeptides described herein are scFv domains, or at least one antigen-binding domain is an scFv domain. DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0092] 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.

[0093] Additional examples and embodiments of antigen binding domains are known in the art.

[0094] Soluble interleukin or cytokine proteins In some embodiments of any of the single-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain can be a soluble interleukin protein, a soluble cytokine protein, or a soluble cell surface 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: 28) aptssstkkt qlqlehllld lqmilnginn yknpkltrml tfkfympkka telkhlqcle eelkpleevl nlaqsknfhl rprdlisnin vivlelkgse ttfmceyade tativeflnr witfcqsiis tlt Human soluble IL-3 (SEQ ID NO: 29) apmtqttplkt swvncsnmid eiithlkqpp lplldfnnln gedqdilmen nlrrpnleaf nravkslqna saiesilknl lpclplataa ptrhpihikd gdwnefrrkl tfylktlena qaqqttlsla if Human soluble IL-7 (SEQ ID NO: 30) dcdiegkdgkqyesv lmvsidqlld smkeigsncl nnefnffkrh icdankegmf lfraarklrq flkmnstgdf dlhllkvseg ttillnctgq vkgrkpaalg eaqptkslee nkslkeqkkl ndlcflkrll qeiktcwnki lmgtkeh Human soluble IL-8 (SEQ ID NO: 31) egavlprsak elrcqcikty skpfhpkfik elrviesgph canteiivkl sdgrelcldp kenwvqrvve kflkraens Human soluble IL-10 (SEQ ID NO: 32) spgqgtqsensc thfpgnlpnm lrdlrdafsr vktffqmkdq ldnlllkesl ledfkgylgc qalsemiqfy leevmpqaen qdpdikahvn slgenlktlr lrlrrchrfl pcenkskave qvknafnklq ekgiykamse fdifinyiea ymtmkirn Human soluble IL-12β (p40) (SEQ ID NO: 33) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS Nucleic acid encoding human soluble IL-12β (p40) (SEQ ID NO: 34) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCCATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACCGATTTAACCTTCTCCGTGAAAAGCAGCCGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTCAGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAAGATAGCGCTTGTCCCGCTGCCGAAGAATCTTTACCCATTGAGGTGATGGTGGACGCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTTATCCGGGACATCATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCAAAAATAGCCGGCAAGTTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACCGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTCC Human soluble IL-12α (p35) (SEQ ID NO: 35) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS ヒトレッックIL-12α(p35)をををでするするするすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすすす. CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAAGATTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGAGCAAAAGAACGAGTCTTGTCCAACTCTCGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTTTATGATGG CTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCACTCCGAGACCGTCCCTCAGAAGTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAAACGCCAGC Exemplary soluble IL-12 IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTD LTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKS KREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLE LTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS Exemplary human soluble IL-12-encoding nucleic acid (SEQ ID NO: 38) Human soluble IL-15 (SEQ ID NO: 39) Nwvnvisdlkki edliqsmhid atlytesdvh psckvtamkc flllelqvisl esgdasihdt venliilann slssngnvte sgckeceele eknikeflqs fvhivqmfin ts Human soluble IL-17 (SEQ ID NO: 40) gitiprn pgcpnsedkn fprtvmvnln ihnrntntnp krssdyynrs tspwnlhrne dperypsviw eakcrhlgci nadgnvdyhm nsvpiqqeil vlrrepphcp nsfrlekilv svgctcvtpi vhhva Human soluble IL-18 (SEQ ID NO: 41) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED Nucleic acid encoding human soluble IL-18 (SEQ ID NO: 42) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACA AGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT Human soluble IL-21 (SEQ ID NO: 43) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS Nucleic acid encoding human soluble IL-21 (SEQ ID NO: 44) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC Human soluble PDGF-DD (SEQ ID NO: 45) rdtsatpqsasi kalrnanlrr desnhltdly rrdetiqvkg ngyvqsprfp nsyprnlllt wrlhsqentr iqlvfdnqfg leeaendicr ydfvevedis etstiirgrw cghkevppri ksrtnqikit fksddyfvak 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: 46) 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: 47) tqdcsfqhspissd favkirelsd yllqdypvtv asnlqdeelc gglwrlvlaq rwmerlktva gskmqgller vnteihfvtk cafqpppscl rfvqtnisrl lqetseqlva lkpwitrqnf srclelqcqp dsstlpppws prpleatapt apqppllllll llpvglllla aawclhwqrt rrrtprpgeq vppvpspqdl llveh

[0095] Exemplary soluble cell surface proteins include soluble MICA, MICB, and ULP16 binding proteins (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, or ULBP6). Exemplary sequences of soluble MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 are listed below. Human soluble MICA (SEQ ID NO: 48) 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: 49) 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: 50) 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: 51) 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: 52) 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: 53) 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: 54) 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: 55) rrddp hslcyditvi pkfrpgprwc avqgqvdekt flhydcgnkt vtpvsplgkk lnvtmawkaq npvlrevvdi lteqlldiql enytpkeplt lqarmsceqk aeghssgswq fsidgqtfll fdsekrmwtt vhpgarkmke kwendkdvam sfhyismgdc igwledflmg mdstlepsag aplamssg

[0096] In some embodiments, the soluble IL-12 protein may comprise a first 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 SEQ ID NO: 33, and a second 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 SEQ ID NO: 35. In some embodiments, the soluble IL-12 may further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first and second sequences.

[0097] In some embodiments, the soluble IL-12 protein is encoded by a first nucleic acid encoding a first 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 SEQ ID NO: 34, and a second nucleic acid sequence that encodes a second 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 SEQ ID NO: 36. In some embodiments, the nucleic acid encoding the soluble IL-12 protein further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first and second nucleic acids.

[0098] In some embodiments, the soluble IL-12 protein comprises a sequence 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 SEQ ID NO: 37. In some embodiments, the soluble IL-12 protein is encoded by a nucleic acid comprising a sequence 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 SEQ ID NO: 38.

[0099] In some embodiments, the soluble IL-18 protein may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 41. In some embodiments, the soluble IL-18 protein is encoded by a nucleic acid that comprises 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 SEQ ID NO: 42.

[0100] In some embodiments, the soluble IL-21 protein may comprise a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 43. In some embodiments, the soluble IL-21 protein is encoded by a nucleic acid comprising a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 44.

[0101] Additional examples of soluble interleukin and cytokine proteins are known in the art.

[0102] Soluble receptors In some embodiments of any of the single-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some embodiments, the soluble receptor is soluble TGF-β receptor II (TGF-βRII) (see, e.g., Yung et al., Am. J. Resp. Crit. Care Med. 194(9):1140-1151, 2016), soluble TGF-βRIII (see, e.g., Heng et al., Placenta 57:320, 2017), or 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).In some embodiments, the soluble cell surface receptor is 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., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389 / fimmu.2018.01150), soluble NKp48 (see, e.g., those described in Mandelboim et al., Nature 409:1055-1060, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018), soluble NKp49 ... 29, 2018; doi:10.3389 / fimmu.2018.01150), soluble DNAM1 (see, e.g., Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389 / fimmu.2018.01150), scMHC1 (see, e.g., Washburn et al., PLoS One 6(3):e18439, 2011), scMHCII (see, e.g., Bishwajit et al., Cellular Immunol. 170(1):25-33, 1996), scTCR (see, e.g., Weber et al., Nature 356(6372):793-796, 1992), soluble CD155 (see, e.g., Tahara-Hanaoka et al., Int. Immunol. 16(4):533-538, 2004), or soluble CD28 (see, e.g., Hebbar et al., Clin. Exp. Immunol. 136:388-392, 2004).

[0103] In some embodiments, the soluble TGFβRII receptor is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 56). ), and a second 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 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 56). In some embodiments, the soluble TGFβRII receptor may further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first and second sequences.

[0104] In some embodiments, the soluble TGFβRII receptor is ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCT At least 70% identical, (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least a first nucleic acid sequence encoding a first sequence that is at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a first nucleic acid sequence encoding a second nucleic acid ... At least 70% identical to TGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (SEQ ID NO: 57), (e.g.,The soluble TGFβRII receptor is encoded by a second nucleic acid sequence that encodes a second sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical). In some embodiments, the nucleic acid encoding the soluble TGFβRII receptor further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first and second nucleic acids.

[0105] In some embodiments, the soluble TGFβRII receptor comprises 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 IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 60). In some embodiments, the soluble TGFβRII receptor is(SEQ ID NO: 61).

[0106] Additional examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0107] Additional antigen-binding domains Some embodiments of any of the single-chain chimeric polypeptides described herein may further comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at their N- and / or C-termini.

[0108] In some embodiments, a single-chain chimeric polypeptide can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus. In some embodiments, one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus of a single-chain chimeric polypeptide can be immediately adjacent to a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein).

[0109] In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at its C-terminus. In some embodiments, one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein).

[0110] In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at its N-terminus and its C-terminus. In some embodiments, one of the one or more additional antigen binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the N-terminus and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains).In some embodiments, one of the one or more additional antigen-binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen-binding domains (e.g., any of the exemplary target binding domains described her...

Claims

1. (i) a first target binding domain; (ii) a soluble tissue factor domain, and (iii) a second target-binding domain A single-chain chimeric polypeptide comprising:

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

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

4. The single-chain chimeric polypeptide of any one of claims 1 to 3, wherein the soluble tissue factor domain and the second target binding domain are immediately adjacent to each other.

5. The single-chain chimeric polypeptide of any one of claims 1 to 3, further comprising a linker sequence between the soluble tissue factor domain and the second target binding domain.

6. The single-chain chimeric polypeptide of any one of claims 1 to 5, wherein the first target-binding domain and the second target-binding domain specifically bind to the same antigen.

7. The single-chain chimeric polypeptide of claim 6 , wherein the first target binding domain and the second target binding domain specifically bind to the same epitope.

8. The single-chain chimeric polypeptide of claim 7 , wherein the first target binding domain and the second target binding domain comprise the same amino acid sequence.

9. The single-chain chimeric polypeptide of any one of claims 1 to 5, wherein the first target-binding domain and the second target-binding domain specifically bind to different antigens.

10. The single-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 is 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 ULBP. 2, 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, PDGFDD, TGF-β receptor II (TGF-βR II) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHC I ligand, scMHC II ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, 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. 11. The single-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, a soluble cytokine protein, or a soluble cell surface protein.

13. 13. The single-chain chimeric polypeptide of claim 12, wherein the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-D, SCF, FLT3L, MICA, MICB, and ULP16 binding protein.

14. 11. The single-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 receptor, a soluble cytokine receptor, or a soluble cell surface receptor.

15. 15. The single-chain chimeric polypeptide of claim 14, wherein the soluble interleukin receptor, soluble cytokine receptor, or soluble cell surface 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.

16. The single-chain chimeric polypeptide of any one of claims 1 to 15, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

17. 17. The single-chain chimeric polypeptide of claim 16, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:

9.

18. The single-chain chimeric polypeptide of any one of claims 1 to 17, further comprising one or more additional target binding domains at its N-terminus and / or C-terminus.

19. A composition comprising any of the single-chain chimeric polypeptides of claims 1 to 18.

20. 20. The composition of claim 19, which is a pharmaceutical composition.

21. 21. A kit comprising at least one dose of the composition of claim 19 or 20.

22. 1. A method for stimulating immune cells, comprising: A method comprising contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides of claims 1 to 18 or the composition of claim 19 or 20.

23. 1. A method for inducing or increasing immune cell proliferation, comprising: A method comprising contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides of claims 1 to 18 or the composition of claim 19 or 20.

24. 1. A method for inducing differentiation of immune cells into memory or memory-like immune cells, comprising: A method comprising contacting an immune cell with an effective amount of any of the single-chain chimeric polypeptides of claims 1 to 18 or the composition of claim 19 or 20.

25. The method of any one of claims 22 to 24, wherein the immune cells are contacted in vitro.

26. The method of any one of claims 22 to 24, wherein the immune cells are contacted in vivo.

27. 27. The method of any one of claims 22 to 26, 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.

28. 28. The method of any one of claims 22 to 27, wherein the immune cells have previously been genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor.

29. The method of any one of claims 22 to 28, further comprising administering the immune cells to a subject in need thereof.

30. 21. A method of 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 single-chain chimeric polypeptides of claims 1-18 or the composition of claim 19 or 20.

31. 21. A method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the single-chain chimeric polypeptides of claims 1-18 or the composition of claim 19 or 20.

32. 32. The method of claim 30 or 31, wherein the subject has been identified or diagnosed as having cancer, an age-related disease or condition, or an infectious disease.

33. 33. The method of claim 32, 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 syndromes (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell carcinoma, gastric esophageal 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.

34. 33. The method of claim 32, 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 decrease in lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, liver cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

35. 33. The method of claim 32, 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.

36. A nucleic acid encoding any of the single-chain chimeric polypeptides of any one of claims 1 to 18.

37. A vector comprising the nucleic acid of claim 36.

38. 38. A cell comprising the nucleic acid of claim 36 or the vector of claim 37.

39. 1. A method for producing a single-chain chimeric polypeptide, comprising: Culturing the cell of claim 38 in a culture medium under conditions sufficient to result in production of the single-chain chimeric polypeptide; recovering said single-chain chimeric polypeptide from said cells and / or said culture medium; A method comprising:

40. 40. A single-chain chimeric polypeptide produced by the method of claim 39.

41. 17. The single-chain chimeric polypeptide of claim 16, wherein the human soluble tissue factor domain does not initiate blood clotting.

42. The single-chain chimeric polypeptide of any one of claims 1 to 18, wherein the soluble tissue factor domain comprises or consists of a sequence derived from wild-type soluble human tissue factor.

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