Single-chain chimeric polypeptides and multi-chain chimeric polypeptides and uses thereof

Abstract

The present disclosure relates to single-chain chimeric polypeptides and multi-chain chimeric polypeptides and uses thereof. The present disclosure relates to the field of biotechnology, and more particularly, to single-chain and multi-chain chimeric polypeptides having a linker domain located between two target binding domains, which can be used in a variety of applications, such as, for example, various target binding domains. Including but not limited to stimulating immune cells, inducing or increasing proliferation of immune cells, inducing differentiation of immune cells, or treating subjects in need thereof (e.g., subjects having cancer or a disease or condition associated with aging).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Patent Application No. 62 / 816,683, filed March 11, 2019; 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 / 881,039, filed July 31, 2019; U.S. Patent Application No. 62 / 724,969, filed August 30, 2018; U.S. Patent Application No. 62 / 817,230, filed March 12, 2019; U.S. Patent Application No. No. 62 / 725,043, filed on August 30, 2018; No. 62 / 725,010, filed on August 30, 2018; No. 62 / 749,007, filed on October 22, 2018; No. 62 / 746,832, filed on October 17, 2018; No. 62 / 749,506, filed on October 23, 2018; No. 62 / 817,241, filed on March 12, 2019; and No. 62 / 881,088, filed on July 31, 2019, each of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present disclosure relates to the field of biotechnology, and more particularly, to single-chain chimeric polypeptides and multi-chain chimeric polypeptides having a linker domain positioned between two target binding domains, which are suitable for various applications. Background Art

[0004] Adoptive immunotherapy or cell therapy requires the in vivo cultivation of immune cells obtained from a subject (and optionally genetic manipulation of the immune cells to express a chimeric antigen receptor or T cell receptor) and then administration back into the subject. A sufficient number of immune cells is required to provide a therapeutic effect in the subject. In many instances, it takes three weeks or more to culture the immune cells obtained from the subject to obtain a therapeutically effective number of immune cells. In addition, many methods for culturing immune cells obtained from a subject in vitro require a layer of feeder cells, which require subsequent purification or separation of the immune cells before administration back into the subject. Summary of the Invention

[0005] The present invention is based on the discovery that single-chain chimeric polypeptides and multi-chain chimeric polypeptides having a linker domain located between two target-binding domains can effectively stimulate immune cells, induce or increase the proliferation of immune cells, induce the differentiation of immune cells, or treat subjects in need thereof (e.g., subjects suffering from cancer or aging-related diseases or conditions). The present invention is also based on the discovery that the multi-chain chimeric polypeptides described herein promote the metabolism of immune cells by increasing their aerobic glycolysis (Warburg effect), oxidative phosphorylation, and mitochondrial reserve respiratory capacity to support their differentiation into effector cells and enhance their effector cell function.

[0006] In some aspects, provided herein are methods for promoting activation and proliferation of natural killer cells or T cells, comprising contacting natural killer cells or T cells in a liquid culture medium under conditions that allow activation and proliferation of natural killer cells or T cells, the liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, wherein the first target binding domain and the second target binding domain are each independently selected from the group consisting of: a soluble interleukin, a soluble cytokine protein or a soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, a soluble cytokine receptor or a soluble cell surface receptor, and a ligand for a co-stimulatory molecule. In some embodiments, the first target binding domain and the linker domain are directly adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain. In some embodiments, the linker domain and the second target binding domain are directly adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the second target binding domain. In some embodiments, the first target binding domain and the second target binding domain are directly adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain. In some embodiments, the second target binding domain and the linker domain are directly 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 linker 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 domains comprise scFv or single domain antibodies.

[0007] In some embodiments of the method of promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain, wherein one or both of the first target binding domain and the second target binding domain bind to a target selected from the group consisting of: CD16a, CD28 , CD3, CD33, CD20, CD19, CD22, CD52, 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, D26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI, ligand of scMHCII, scTC R 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.

[0008] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, wherein one or both of the first target binding domain and the second target binding domain are soluble interleukins or cytokine proteins. 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 are soluble interleukins or cytokine receptors. In some embodiments, soluble interleukins or cytokine receptors are soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155 or soluble CD28. In some embodiments, one or both of the first target binding domain and the second target binding domain are ligands of costimulatory molecules. In some embodiments, the ligands of costimulatory molecules are soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d or LLT-1.

[0009] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, wherein the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind to Factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the multi-single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

[0010] In some embodiments of the method for promoting the activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium containing an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, wherein the IgG1 antibody construct includes at least one antigen binding domain that specifically binds to a soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of a kappa chain and a lambda chain. In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen binding domains in the monoclonal IgG1 antibody specifically bind to the linker domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen binding domains in the bispecific IgG1 antibody specifically binds to the linker domain. 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 directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domains and the first target binding domain, the second target binding domain, or the linker 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 directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the at least one additional target binding domains and the first target binding domain, the second target binding domain, or the linker 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 target binding domains at the N-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target binding domains at the N-terminus and the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, one of the one or more additional target binding domains at the C-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the linker domain.In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target binding domains and the first target binding domain, the second target binding domain, or the linker domain at the C-terminus.

[0011] In some embodiments of the method for promoting the activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, wherein two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. 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 other 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 other 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 other 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 other target binding domains are each antigen binding domains. In some embodiments, the antigen binding domain comprises an scFv or a single domain antibody.

[0012] In some embodiments of the method of promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain, wherein one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a selected group consisting of Targets: 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, CTL A4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4ACMUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI, ligand of scMHCII, s cTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor and CD28 receptor. In some embodiments, one or more of the first target binding domain, the second target binding domain and the one or more additional target binding domains are soluble interleukins, soluble cytokine proteins or soluble cell surface proteins.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, 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 interleukin or cytokine receptor is a soluble TGF-β receptor II (TGFβRII), a soluble TGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, scMHCI, scMHCII, scTCR, a soluble CD155, a soluble CD122, a soluble CD3 or a soluble CD28. In some embodiments, one or more of the first target binding domain, the second target binding domain, and one or more other target binding domains are ligands of costimulatory molecules. In some embodiments, the ligand of the costimulatory molecule is soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d or LLT-1.

[0013] In some embodiments of the method for promoting the activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, the single-chain chimeric polypeptide further comprising a signal sequence at its N-terminus. In some embodiments, the single-chain chimeric polypeptide lacks a signal sequence at its N-terminus. In some embodiments, the single-chain chimeric polypeptide further comprises a peptide tag located at the N-terminus or C-terminus of the single-chain chimeric polypeptide. 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 lacks a signal sequence at its N-terminus.

[0014] In some embodiments of the method for promoting the activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, the contacting step being performed for a period of about 2 hours to about 20 days. In some embodiments, the contacting step is performed for a period of about 1 day to about 15 days. In some embodiments, the liquid culture medium is a serum-free liquid culture medium. In some embodiments, the liquid culture medium is a chemically defined liquid culture medium. In some embodiments, the liquid culture medium comprises the single-chain chimeric polypeptide and the IgG1 antibody construct at a molar ratio of about 0.5:1 to about 2:1. In some embodiments, the liquid culture medium comprises the single-chain chimeric polypeptide and the IgG1 antibody construct at a molar ratio of about 0.8:1 to about 1.2:1.

[0015] In some embodiments of the method for promoting the activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, the NK cells or T cells being previously obtained from a subject. In some embodiments, the method further comprises obtaining NK cells or T cells from a subject before the contacting step. In some embodiments, the NK cells or T 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 a chimeric antigen receptor or a recombinant T cell receptor into the NK cells or T cells. In some embodiments, the method further comprises, before the contacting step, introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells or T cells. In some embodiments, the method further comprises, after the contacting step, isolating the NK cells or T cells. In some embodiments, after the contacting step, the expression level or secretion of one or more proteins selected from the group consisting of TNF-α, IFN-γ, granzyme A, granzyme B, perforin, 2B4, CD8, CD11a, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, ... CD158a, CD158b, CD158e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-1, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, K IR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-1, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-α and IFN-γ.

[0016] In some embodiments of the method of promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain, the method further comprising administering the NK cells or T cells to a subject in need thereof after the contacting step. In some embodiments, the subject has been identified or diagnosed 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, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, 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. In some embodiments, the subject has been identified or diagnosed as having cancer. In some embodiments, the cancer is selected from the group consisting of: a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a Hodgkin's lymphoma, a chronic lymphocytic leukemia (CLL), an acute myeloid leukemia (AML), a chronic myeloid leukemia (CML), an acute lymphocytic leukemia (ALL), a myelodysplastic syndrome (MDS), a cutaneous T-cell lymphoma, a retinoblastoma, a gastric cancer, an urothelial cancer, a lung cancer, a renal cell carcinoma, a gastroesophageal cancer, a pancreatic cancer, a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a non-small cell lung cancer, a head and neck squamous cell carcinoma, an endometrial cancer, a cervical cancer, a liver cancer, and a hepatocellular carcinoma. In some embodiments, the subject has been diagnosed or identified 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, hepatitis A and C viruses, papillomavirus, and influenza virus.

[0017] In some aspects, provided herein are activated NK cells or T cells produced by any of the methods described herein, the method using a single-chain chimeric polypeptide. In some aspects, provided herein are pharmaceutical compositions comprising such activated NK cells or T cells. In some aspects, provided herein are kits comprising such pharmaceutical compositions. In some aspects, provided herein are methods for killing cancer cells, infected cells, or senescent cells in subjects in need, comprising administering to a subject a therapeutically effective amount of activated NK cells or activated T cells produced by any of the methods described herein, or a pharmaceutical composition comprising such NK cells or T cells, the method using a single-chain chimeric polypeptide. In certain embodiments, the subject has been identified or diagnosed as suffering from cancer. In some embodiments, the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a 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, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having a disease or condition associated with aging. In some embodiments, the disease or condition associated with aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, 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.

[0018] In some embodiments, a method of killing cancer cells, infected cells, or senescent cells in a subject in need thereof comprises administering to the subject a therapeutically effective amount of activated NK cells or activated T cells produced by any of the methods described herein, or a pharmaceutical composition comprising such activated NK cells or T cells, wherein the production method employs a single-chain chimeric polypeptide. In some embodiments, the subject has been identified or diagnosed as having cancer. In some embodiments, the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a 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, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having a disease or condition associated with aging. In certain embodiments, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to aging.In certain embodiments, the disease or the patient's condition relevant to aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, myocardial cell hypertrophy, osteoarthritis, Parkinson's disease, age-related lung tissue elasticity loss, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis and renal dysfunction.In certain embodiments, the subject has been diagnosed or identified as suffering from 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, hepatitis A and C viruses, papillomavirus, and influenza virus.

[0019] In some aspects, a kit is provided herein comprising: (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, wherein the first target binding domain and the second target binding domain are each independently selected from the group consisting of: a soluble interleukin or cytokine protein, an antigen binding domain, a soluble interleukin or cytokine receptor, and a ligand of a co-stimulatory molecule; and (ii) an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain. In some embodiments, the first target binding domain and the linker domain are directly adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain. In some embodiments, the linker domain and the second target binding domain are directly adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the second target binding domain. In some embodiments, the first target binding domain and the second target binding domain are directly adjacent to each other. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain. In some embodiments, the second target binding domain and the linker domain are directly 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 linker 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.

[0020] In some embodiments of the kit comprising a single-chain chimeric polypeptide and an IgG1 antibody construct, one or both of the first target binding domain and the second target binding domain binds to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VIST A. CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EP CAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL In some embodiments, one or both of the first target binding domain and the second target binding domain are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins. 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 are soluble interleukin receptors, soluble cytokine receptors or soluble cell surface receptors. In some embodiments, soluble interleukin receptors, soluble cytokine receptors or soluble cell surface receptors are soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155 or soluble CD28. In some embodiments, one or both of the first target binding domain and the second target binding domain are ligands of costimulatory molecules. In some embodiments, the ligand of the costimulatory molecule is soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CDld, or LLT-1.

[0021] In some embodiments of the kit comprising a single-chain chimeric polypeptide and an IgG1 antibody construct, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind to Factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the single-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

[0022] In some embodiments of the kit comprising a single-chain chimeric polypeptide and an IgG1 antibody construct, the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to a soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of a kappa chain and a lambda chain. In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains in the monoclonal IgG1 antibody specifically bind to the linker domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains in the bispecific IgG1 antibody specifically binds to the linker domain.

[0023] In some embodiments of the kit comprising a single-chain chimeric polypeptide and an IgG1 antibody construct, 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 directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between at least one of the additional target binding domains and the first target binding domain, the second target binding domain, or the linker 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 directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between at least one of the additional target binding domains and the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide comprises one or more additional target binding domains at both its N-terminus and its C-terminus. In some embodiments, one of the one or more additional target binding domains at the N-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target binding domains at the N-terminus and the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, one of the one or more additional target binding domains at the C-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target binding domains at the C-terminus and the first target binding domain, the second target binding domain, or the linker domain.

[0024] In some embodiments of the kit comprising a single-chain chimeric polypeptide and an IgG1 antibody construct, two or more of the first target binding domain, the second target binding domain, and one or more other 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 one or more other 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 one or more other target binding domains comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and one or more other target binding domains each specifically bind to the same antigen. In some embodiments, the first target binding domain, the second target binding domain, and one or more other target binding domains each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and one or more other target binding domains each comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and one or more other 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 other target binding domains are each antigen binding domains. In some embodiments, the antigen binding domain comprises an scFv or a single domain antibody.

[0025] In some embodiments of the kit comprising a single-chain chimeric polypeptide and an IgG1 antibody construct, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MIC B. IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA , B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI, ligand of scMHCII, ligand of scTCR In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins.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 more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some embodiments, the soluble 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, scMHCI, scMHCII, scTCR, a soluble CD155, a soluble CD122, a soluble CD3 or a soluble CD28. In some embodiments, one or both of the first target binding domain and the second target binding domain are ligands of a co-stimulatory molecule. In some embodiments, the ligand of the co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d or LLT-1. In some embodiments, the single-chain chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the single-chain chimeric polypeptide. 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 lacks a signal sequence at its N-terminus.

[0026] In some aspects, provided herein are methods for promoting activation and proliferation of natural killer cells or T cells, comprising contacting the natural killer cells or T cells in a liquid culture medium under conditions permissive for activation and proliferation of the natural killer cells or T cells, the liquid culture medium comprising: (1) an effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain. In some embodiments, the first target binding domain and the linker domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the first target-binding domain and the linker domain in the first chimeric polypeptide. In some embodiments, the linker domain and the first domain of a pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the linker domain in the first chimeric polypeptide and the first domain of a pair of affinity domains. In some embodiments, the second domain of a pair of affinity domains and the second target-binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between the second domain of a pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. In some embodiments, the first target-binding domain and the second target-binding domain specifically bind to the same antigen. In some embodiments, the first target-binding domain and the second target-binding domain specifically bind to the same epitope. In some embodiments, the first target-binding domain and the second target-binding domain comprise the same amino acid sequence. In some embodiments, the first target-binding domain and the second target-binding domain specifically bind to different antigens. In some embodiments, one or both of the first target-binding domain and the second target-binding domain are antigen-binding domains. In some embodiments, the first target binding domain and the second target binding domain are each an antigen binding domain. In some embodiments, the antigen binding domain comprises a scFv or a single domain antibody.

[0027] In some embodiments of the method of promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting natural killer cells or T cells in a liquid culture medium, wherein the liquid culture medium comprises a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct, wherein the IgG1 antibody construct comprises at least one antigen binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, and one or both of the first target binding domain and the second target binding domain specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD12 3. 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, C D200, CD80, CD86, PD-L2, B7-H4HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, M UC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, scMHC In some embodiments, the first target binding domain and the second target binding domain are 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, stem cell factor (SCF), stem cell-like tyrosine kinase 3 ligand (FLT3L), MICA, MICB, ULP16 binding protein, CD155, CD122, and CD28. In some embodiments, one or both of the first target binding domain and the second target binding domain are soluble interleukins or cytokine proteins. One or both of the first target binding domain and the second target binding domain are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins.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 are soluble interleukin receptors, soluble cytokine receptors, or soluble cell surface receptors. In some embodiments, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28. In some embodiments, one or both of the first target binding domain and the second target binding domain is a ligand of a co-stimulatory molecule. In some embodiments, the ligand of the co-stimulatory molecule is soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d or LLT-1.

[0028] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium, wherein the liquid culture medium comprises a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct, wherein the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, the first chimeric polypeptide further comprising one or more additional target-binding domains, wherein at least one of the one or more target-binding domains is located between the linker domain and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the linker domain and at least one of the one or more target antigen-binding domains, and / or a linker sequence between at least one of the one or more target antigen-binding domains and the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises one or more additional target-binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target-binding domains is directly adjacent to the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of a pair of affinity domains. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain. In some embodiments, at least one of the one or more additional target binding domains is positioned at the N-terminus and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains is positioned between the linker domain in the first chimeric polypeptide and the first domain of a pair of affinity domains. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains positioned at the N-terminus is directly adjacent to the first target binding domain or the first domain of a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of a pair of affinity domains. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains disposed at the C-terminus is directly adjacent to the first target binding domain or the first domain of a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed between the at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of a pair of affinity domains.In some embodiments, at least one of the one or more additional target binding domains positioned between the linker domain and the first domain of a pair of affinity domains is directly adjacent to the linker domain and / or the first domain of a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence positioned: (i) between the linker domain and at least one of the one or more additional target binding domains that are positioned between the linker domain and the first domain of a pair of affinity domains, and / or (ii) between the first domain of a pair of affinity domains and at least one of the one or more additional target binding domains that are positioned between the linker domain and the first domain of a pair of affinity domains.

[0029] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium, wherein the liquid culture medium comprises a first chimeric polypeptide and a second chimeric polypeptide, and an IgG1 antibody construct, wherein the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, the second chimeric polypeptide further comprising one or more additional target-binding domains at the N-terminus and / or C-terminus of the second chimeric polypeptide. In some embodiments, in the second chimeric polypeptide, at least one of the one or more additional target-binding domains is directly adjacent to the second domain of a pair of affinity domains. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains in the second chimeric polypeptide and the second domain of the pair of affinity domains. In some embodiments, in the second chimeric polypeptide, at least one of the one or more additional target-binding domains is directly adjacent to the second target-binding domain. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target-binding domains in the second chimeric polypeptide and the second target-binding domain.

[0030] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, wherein two or more of the first target-binding domain, the second target-binding domain, and the one or more additional target-binding domains specifically bind to the same antigen. 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 other 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 other 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 other target binding domains are each antigen binding domains. In some embodiments, the antigen binding domain comprises an scFv.

[0031] In some embodiments of the method of promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting natural killer cells or T cells in a liquid culture medium, the liquid culture medium comprising a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct, the IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains that specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD 22. 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, C D30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4A C, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins.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 more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some embodiments, the soluble 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, scMHCI, scMHCII, scTCR, a soluble CD155, a soluble CD122, a soluble CD3, or a soluble CD28. In some embodiments, the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide. One or both of the first target binding domain and the second target binding domain are ligands of a co-stimulatory molecule. In some embodiments, the ligand of the co-stimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d, or LLT-1. In some embodiments, the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide.

[0032] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, the linker domain being a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind to Factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

[0033] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium, wherein the liquid culture medium comprises a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct, wherein the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, and the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to a soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of a kappa chain and a lambda chain. In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains in the monoclonal IgG1 antibody specifically bind to the linker domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains in the bispecific IgG1 antibody specifically binds to the linker domain.

[0034] In some embodiments of the method for promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, wherein the pair of affinity domains are a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. In some embodiments, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments, the human IL15Rα is mature, full-length IL15Rα. In some embodiments, the pair of affinity domains is selected from the group consisting of barnase and barnstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins synaptobrevin, synaptotagmin, synaptophysin, and SNAP25. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide lacks a signal sequence at its N-terminus.

[0035] In some embodiments of the method for promoting activation and proliferation of natural killer (NK) cells or T cells, the method comprises contacting the natural killer (NK) cells or T cells in a liquid culture medium comprising a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, the contacting step being performed for a period of about 2 hours to about 20 days. In some embodiments, the contacting step is performed for a period of about 1 day to about 15 days. In some embodiments, the liquid culture medium is a serum-free liquid culture medium. In some embodiments, the liquid culture medium is a chemically defined liquid culture medium. In some embodiments, the liquid culture medium comprises the multi-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of about 0.5:1 to about 2:1. In some embodiments, the liquid culture medium comprises the multi-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of about 0.8:1 to about 1.2:1. In some embodiments, the NK cells or T cells are previously obtained from a subject. In some embodiments, the method further comprises obtaining the NK cells or T cells from the subject prior to the contacting step. In some embodiments, the NK cells or T cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor. In some embodiments, the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cell or T cell after the contacting step. In some embodiments, the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cell or T cell before the contacting step. In some embodiments, the method further comprises isolating the NK cell or T cell after the contacting step. In some embodiments, the expression level or secretion of one or more proteins of the NK cell or T cell is increased after the contacting step compared to the expression level or secretion of one or more proteins before the contacting step, the protein being selected from the group consisting of: TNF-α, IFN-γ, granzyme A, granzyme B, perforin, 2B4, CD8, CD11a, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CD158a, CD158b, CD158e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-1, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, K IR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-1, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-α and IFN-γ.In some embodiments, the method further comprises administering NK cells or T cells to a subject in need thereof after the contacting step.

[0036] In some embodiments of the method of promoting activation and proliferation of natural killer cells or T cells, the method comprises contacting the natural killer cells or T cells in a liquid culture medium comprising a first chimeric polypeptide and a second chimeric polypeptide and an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to a linker domain in one of the chimeric polypeptides, and the subject has been identified or diagnosed 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, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, 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. In some embodiments, the subject has been identified or diagnosed as having cancer. In some embodiments, the cancer is selected from the group consisting of: a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a Hodgkin's lymphoma, a chronic lymphocytic leukemia (CLL), an acute myeloid leukemia (AML), a chronic myeloid leukemia (CML), an acute lymphocytic leukemia (ALL), a myelodysplastic syndrome (MDS), a cutaneous T-cell lymphoma, a retinoblastoma, a gastric cancer, an urothelial cancer, a lung cancer, a renal cell carcinoma, a gastroesophageal cancer, a pancreatic cancer, a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a non-small cell lung cancer, a head and neck squamous cell carcinoma, an endometrial cancer, a cervical cancer, a liver cancer, and a hepatocellular carcinoma. In some embodiments, the subject has been diagnosed or identified 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, hepatitis A and C viruses, papillomavirus, and influenza virus.

[0037] In some aspects, provided herein are activated NK cells or T cells produced by any of the methods described herein, the method using multi-chain chimeric polypeptides. In some aspects, provided herein are pharmaceutical compositions comprising such activated NK cells or T cells. In some aspects, provided herein are kits comprising such pharmaceutical compositions. In some aspects, provided herein are methods for killing cancer cells, infected cells, or senescent cells in subjects in need, comprising administering to subjects a therapeutically effective amount of activated NK cells or activated T cells produced by any of the methods described herein, or a pharmaceutical composition comprising such NK cells or T cells, the method using multi-chain chimeric polypeptides. In certain embodiments, subjects have been identified or diagnosed as suffering from cancer. In some embodiments, the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a 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, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having a disease or condition associated with aging. In some embodiments, the disease or condition associated with aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, 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.

[0038] In some embodiments, a method of killing cancer cells, infected cells, or senescent cells in a subject in need thereof comprises administering to the subject a therapeutically effective amount of activated NK cells or activated T cells produced by any of the methods described herein, or a pharmaceutical composition comprising such activated NK cells or T cells, wherein the production method employs a multi-chain chimeric polypeptide. In some embodiments, the subject has been identified or diagnosed as having cancer. In some embodiments, the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a 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, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed as having a disease or condition associated with aging. In certain embodiments, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to aging.In certain embodiments, the disease or the patient's condition relevant to aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, myocardial cell hypertrophy, osteoarthritis, Parkinson's disease, age-related lung tissue elasticity loss, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis and renal dysfunction.In certain embodiments, the subject has been diagnosed or identified as suffering from 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, hepatitis A and C viruses, papillomavirus, and influenza virus.

[0039] In some aspects, provided herein are kits comprising: 1) a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain. In some embodiments, the first target binding domain and the linker domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain in the first chimeric polypeptide. In some embodiments, the linker domain and the first domain of the pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the linker domain in the first chimeric polypeptide and the first domain in the pair of affinity domains. In some embodiments, the second domain in the pair of affinity domains and the second target-binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between the second domain in the pair of affinity domains and the second target-binding domain in the second chimeric polypeptide. In some embodiments, the first target-binding domain and the second target-binding domain specifically bind to the same antigen. In some embodiments, the first target-binding domain and the second target-binding domain specifically bind to the same epitope. In some embodiments, the first target-binding domain and the second target-binding domain comprise the same amino acid sequence. In some embodiments, the first target-binding domain and the second target-binding domain specifically bind to different antigens. In some embodiments, one or both of the first target-binding domain and the second target-binding domain are antigen-binding domains. In some embodiments, the first target-binding domain and the second target-binding domain are each antigen-binding domains. In some embodiments, the antigen-binding domain comprises an scFv or a single-domain antibody.

[0040] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, one or both of the first target binding domain and the second target binding domain specifically binds to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL- 8. TNFα, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VI STA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, E PCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI, ligand of scMHCII, ligand of scTCR, IL In some embodiments, one or both of the first target binding domain and the second target binding domain are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins. 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 are soluble interleukin receptors, soluble cytokine receptors or soluble cell surface receptors. In some embodiments, soluble receptors are soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155 or soluble CD28. In some embodiments, one or both of the first target binding domain and the second target binding domain are ligands of costimulatory molecules. In some embodiments, the ligands of costimulatory molecules are soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d or LLT-1.

[0041] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more target binding domains is located between the linker domain and the first domain of a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the linker domain and at least one of the one or more target antigen binding domains, and / or a linker sequence between at least one of the one or more target antigen binding domains and the first domain of a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the first domain of a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of a pair of affinity domains. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain. In some embodiments, at least one of the one or more additional target binding domains is positioned at the N-terminus and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains is positioned between the linker domain in the first chimeric polypeptide and the first domain in a pair of affinity domains. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains positioned at the N-terminus is directly adjacent to the first target binding domain or the first domain in a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain or the first domain in a pair of affinity domains. In some embodiments, in the first chimeric polypeptide, at least one of the one or more additional target binding domains positioned at the C-terminus is directly adjacent to the first target binding domain or the first domain in a pair of affinity domains. In some embodiments, the first chimeric polypeptide further comprises a linker sequence positioned between at least one additional target binding domain and the first target binding domain or the first domain of a pair of affinity domains in the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains positioned between the linker domain and the first domain of a pair of affinity domains is directly adjacent to the linker domain and / or the first domain of a pair of affinity domains.In some embodiments, the first chimeric polypeptide further comprises a linker sequence positioned: (i) between the linker domain and at least one of the one or more additional target binding domains that are positioned between the linker domain and the first domain of a pair of affinity domains, and / or (ii) between the first domain of a pair of affinity domains and at least one of the one or more additional target binding domains that are positioned between the linker domain and the first domain of a pair of affinity domains.

[0042] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide. In some embodiments, in the second chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the second domain of a pair of affinity domains. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain of a pair of affinity domains. In some embodiments, in the second chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the second target binding domain. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

[0043] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, two or more of the first target binding domain, the second target binding domain, and the one or more other 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 other target binding domains specifically bind to the same epitope. Two or more of the first target binding domain, the second target binding domain, and the one or more other 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 other 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 other 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 other 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 other 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 other target binding domains are each antigen binding domains. In some embodiments, the antigen binding domain comprises an scFv.

[0044] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD2 72. VISTA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3 , EPCAM, BCMA, P-cadherin, CEACAM5, UL16-binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI, ligand of scMHCII, ligand of scTCR, IL-1 In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins.In some embodiments, the soluble interleukin, soluble cytokine protein, or soluble cell surface receptor 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 is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some embodiments, the soluble receptor is a soluble TGF-β receptor II (TGFβRII), a soluble TGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, scMHCI, scMHCII, scTCR, a soluble CD155, a soluble CD122, a soluble CD3 or a soluble CD28. In some embodiments, one or both of the first target binding domain and the second target binding domain are ligands of a costimulatory molecule. In some embodiments, the ligand of the costimulatory molecule is a soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d or LLT-1.

[0045] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 1. In some embodiments, the soluble human tissue factor domain does not include one or more of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind to Factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments, the multi-chain chimeric polypeptide does not stimulate blood coagulation in a mammal.

[0046] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to a soluble tissue factor domain. In some embodiments, the linker domain is selected from the group consisting of a kappa chain and a lambda chain. In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains in the monoclonal IgG1 antibody specifically bind to the linker domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains in the bispecific IgG1 antibody specifically binds to the linker domain.

[0047] In some embodiments of the kit comprising a multi-chain chimeric polypeptide and an IgG1 antibody construct, the pair of affinity domains is a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. In some embodiments, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments, the human IL15Rα is a mature full-length IL15Rα. In some embodiments, the pair of affinity domains is selected from the group consisting of: barnase and barnstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins synaptobrevin, synaptotagmin, synaptophysin, and SNAP25. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide lacks a signal sequence at its N-terminus. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide lacks a signal sequence at its N-terminus.

[0048] Also provided herein is a method for increasing glucose consumption by immune cells, comprising: contacting the immune cells in a liquid culture medium under conditions that allow glucose consumption by the immune cells, wherein the liquid culture medium comprises an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and optionally (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain, wherein the first target binding domain and the second target binding domain are each independently selected from the following group: a soluble interleukin, a soluble cytokine protein or a soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, a soluble cytokine receptor or a soluble cell surface receptor, and a ligand of a co-stimulatory molecule.

[0049] Also provided herein is a method for increasing oxidative phosphorylation in an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow oxidative phosphorylation of the immune cell, wherein the liquid culture medium comprises an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and optionally (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain, wherein the first target binding domain and the second target binding domain are each independently selected from the following group: a soluble interleukin, a soluble cytokine protein or a soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, a soluble cytokine receptor or a soluble cell surface receptor, and a ligand of a co-stimulatory molecule.

[0050] Also provided herein is a method for increasing aerobic glycolysis in an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow aerobic glycolysis in the immune cell, wherein the liquid culture medium comprises an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and optionally (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain, wherein the first target binding domain and the second target binding domain are each independently selected from the group consisting of a soluble interleukin, a soluble cytokine protein or a soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, a soluble cytokine receptor or a soluble cell surface receptor, and a ligand for a co-stimulatory molecule.

[0051] Also provided herein is a method for increasing the extracellular acidification rate (ECAR) of an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow extracellular acidification of the immune cell, wherein the liquid culture medium comprises an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and optionally (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain, wherein the first target binding domain and the second target binding domain are each independently selected from the following group: a soluble interleukin, a soluble cytokine protein or a soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, a soluble cytokine receptor or a soluble cell surface receptor, and a ligand of a co-stimulatory molecule.

[0052] Also provided herein is a method for increasing the mitochondrial oxygen consumption rate of an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow the mitochondrial oxygen consumption rate of the immune cell, wherein the liquid culture medium comprises an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and optionally (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain, wherein the first target binding domain and the second target binding domain are each independently selected from the following group: a soluble interleukin, a soluble cytokine protein or a soluble cell surface protein, an antigen binding domain, a soluble interleukin receptor, a soluble cytokine receptor or a soluble cell surface receptor, and a ligand of a co-stimulatory molecule.

[0053] In some embodiments of any of the methods described herein, the liquid culture medium comprises a single-chain chimeric polypeptide and an IgG1 antibody construct. In some embodiments of any of the methods described herein, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains in the monoclonal IgG1 antibody specifically bind to a linker domain. In some embodiments of any of the methods described herein, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains in the bispecific IgG1 antibody specifically binds to a linker domain.

[0054] In some embodiments of any of the methods described herein, the contacting step is carried out for a period of time from about 2 hours to about 20 days (e.g., from about 1 day to about 15 days). In some embodiments of any of the methods described herein, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically defined liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium includes serum. In some embodiments of any of the methods described herein, the liquid culture medium includes the single-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of about 0.5:1 to about 2:1 (e.g., from about 0.8:1 to about 1.2:1).

[0055] In some embodiments of any of the methods described herein, the first target binding domain and the linker domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain. In some embodiments of any of the methods described herein, the linker domain and the second target binding domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the second target binding domain. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are directly adjacent to each other.

[0056] In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the second target binding domain. In some embodiments of any of the methods described herein, the second target binding domain and the linker domain are directly adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the second target binding domain and the linker domain.

[0057] In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0058] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv or a single domain antibody.

[0059] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain binds to a target selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD52, 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, CD 26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD13 7. CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, B CMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand of TGF-β receptor II (TGF-βRII), ligand of TGF-βRIII, ligand of DNAM-1, ligand of NKp46, ligand of NKp44, ligand of NKG2D, ligand of NKp30, ligand of scMHCI, ligand of scMHCII, ligand of scTCR, ligand 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-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.

[0060] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine protein, or a 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.

[0061] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain are soluble interleukin receptors, soluble cytokine receptors, or soluble cell surface receptors. In some embodiments of any of the methods described herein, the soluble interleukin receptors, soluble cytokine receptors, or soluble cell surface receptors are 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.

[0062] In some embodiments of any of the methods described herein, the linker domain is a soluble tissue factor domain. In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence that is at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1. In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include one or more of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein.

[0063] In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include any of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein.

[0064] In some embodiments of any of the methods described herein, the soluble tissue factor domain is not capable of binding to Factor VIIa. In some embodiments of any of the methods described herein, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide does not stimulate blood coagulation in mammals. In some embodiments of any of the methods described herein, the IgG1 antibody construct includes at least one antigen binding domain that specifically binds to the soluble tissue factor domain. In some embodiments of any of the methods described herein, the linker domain is selected from the following groups: kappa chain and lambda chain. In some embodiments of any of the methods described herein, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen binding domains in the monoclonal IgG1 antibody specifically bind to the linker domain. In some embodiments of any of the methods described herein, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen binding domains in the bispecific IgG1 antibody specifically binds to the linker domain.

[0065] In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and / or C-terminus. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus. In some embodiments of any of the methods described herein, the one or more additional target binding domains are directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between at least one of the additional target binding domains and the first target binding domain, the second target binding domain, or the linker domain.

[0066] In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its C-terminus. In some embodiments of any of the methods described herein, one of the one or more additional target binding domains is directly adjacent to the first target binding domain, the second target binding domain, or a linker domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between at least one of the additional target binding domains and the first target binding domain, the second target binding domain, or the linker domain.

[0067] In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide comprises one or more additional target binding domains at its N-terminus and C-terminus. In some embodiments of any of the methods described herein, one of the one or more additional target binding domains at the N-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target binding domains at the N-terminus and the first target binding domain, the second target binding domain, or the linker domain. In some embodiments of any of the methods described herein, one of the one or more additional target binding domains at the C-terminus is directly adjacent to the first target binding domain, the second target binding domain, or the linker domain. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between one of the one or more additional target binding domains at the C-terminus and the first target binding domain, the second target binding domain, or the linker domain.

[0068] In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to different antigens.

[0069] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more other target binding domains are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv or a single domain antibody.

[0070] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of: CD16a, 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, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VIS TA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EP CAM, BCMA, P-cadherin, CEACAM5, aUL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL -1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor and CD28 receptor.

[0071] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins. In some embodiments of any of the methods described herein, the soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins are 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.

[0072] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more other target binding domains are soluble interleukin receptors, soluble cytokine receptors, or soluble cell surface receptors. In some embodiments of any of the methods described herein, the soluble interleukins, soluble cytokine receptors, or soluble cell surface receptors are soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.

[0073] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains are ligands of costimulatory molecules. In some embodiments of any of the methods described herein, the ligand of the costimulatory molecule is soluble CD80, CD86, CD40, ICOSL, CD70, OX40L, 4-1BBL, GITRL, LIGHT, TIM3, TIM4, ICAM1, LFA3, CD1d, or LLT-1.

[0074] In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the single-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the immune cells are previously obtained from the subject. Some embodiments of any of the methods described herein further comprise obtaining immune cells from the subject before the contacting step. In some embodiments of any of the methods described herein, the immune cells have previously been genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor. Some embodiments of any of the methods described herein further comprise introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the immune cells after the contacting step. Some embodiments of any of the methods described herein further comprise introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the immune cells before the contacting step. Some embodiments of any of the methods described herein further comprise isolating the immune cells after the contacting step. In some embodiments of any of the methods described herein, after the contacting step, the immune cells express or secrete increased levels of one or more proteins selected from the group consisting of TNF-α, IFN-γ, granzyme A, granzyme B, perforin, 2B4, CD8, CD11a, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94 ... 37, CD158a, CD158b, CD158e, CD178, CD226, CD253, NKG2A, NKG2C, NKG2D, LIR-1, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-1, NKG2A, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-α, and IFN-γ. The immune cells are administered to a subject in need thereof.

[0075] In some embodiments of any method described herein, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to age. In some embodiments of any method described herein, the disease or the patient's condition relevant to age is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, myocardial cell hypertrophy, osteoarthritis, Parkinson's disease, loss of lung tissue elasticity relevant to age, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

[0076] In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing's sarcomas, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-Hodgkin's lymphomas, Hodgkin's lymphomas, 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, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

[0077] In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an infectious disease. In some embodiments of any of the methods described herein, 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, or influenza virus.

[0078] Also provided herein are activated immune cells produced by any of the methods described herein. Also provided herein are pharmaceutical compositions comprising any of the activated immune cells produced by any of the methods described herein. Also provided herein are kits comprising pharmaceutical compositions comprising any of the activated immune cells described herein produced by any of the methods described herein.

[0079] Also provided herein are methods of killing cancer cells, infected cells, or senescent cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced by any of the methods described herein or any of the pharmaceutical compositions described herein. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a Hodgkin's lymphoma, a chronic lymphocytic leukemia (CLL), an acute myeloid leukemia (AML), a chronic myeloid leukemia (CML), an acute lymphocytic leukemia (ALL), a myelodysplastic syndrome (MDS), a cutaneous T-cell lymphoma, a retinoblastoma, a gastric cancer, an urothelial cancer, a lung cancer, a renal cell carcinoma, a gastroesophageal cancer, a pancreatic cancer, a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a non-small cell lung cancer, a head and neck squamous cell carcinoma, an endometrial cancer, a cervical cancer, a liver cancer, and a hepatocellular carcinoma.

[0080] In some embodiments of any method described herein, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to aging. In some embodiments of any method described herein, the disease or the patient's condition relevant to aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related 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.

[0081] Also provided herein are methods of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any activated immune cell described herein produced by any of the methods described herein or any pharmaceutical composition described herein. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a Hodgkin's lymphoma, a chronic lymphocytic leukemia (CLL), an acute myeloid leukemia (AML), a chronic myeloid leukemia (CML), an acute lymphocytic leukemia (ALL), a myelodysplastic syndrome (MDS), a cutaneous T-cell lymphoma, a retinoblastoma, a gastric cancer, an urothelial cancer, a lung cancer, a renal cell carcinoma, a gastroesophageal cancer, a pancreatic cancer, a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a non-small cell lung cancer, a head and neck squamous cell carcinoma, an endometrial cancer, a cervical cancer, a liver cancer, and a hepatocellular carcinoma.

[0082] In some embodiments of any method described herein, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to aging. In some embodiments of any method described herein, the disease or the patient's condition relevant to aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related 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.

[0083] In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an infectious disease. In some embodiments of any of the methods described herein, 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, or influenza virus.

[0084] Also provided herein is a method for increasing glucose consumption by an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow glucose consumption by the immune cell, the liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide, the first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide, the second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct, the IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain.

[0085] Also provided herein is a method for increasing oxidative phosphorylation in an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions permissive for oxidative phosphorylation of the immune cell, the liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain.

[0086] Also provided herein is a method for increasing aerobic glycolysis in an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow aerobic glycolysis in the immune cell, the liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide, the first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide, the second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct, the IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain.

[0087] Also provided herein is a method for increasing the extracellular acidification rate (ECAR) of an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow extracellular acidification of the immune cell, the liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide, the first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide, the second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct, the IgG1 antibody construct comprising at least one antigen binding domain that specifically binds to the linker domain.

[0088] Also provided herein is a method for increasing the mitochondrial oxygen consumption rate of an immune cell, comprising: contacting the immune cell in a liquid culture medium under conditions that allow the mitochondrial oxygen consumption rate of the immune cell, the liquid culture medium comprising (1) an effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide, the first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; (b) a second chimeric polypeptide, the second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated through the binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct, the IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain.

[0089] In some embodiments of any method described herein, liquid culture medium includes single-chain chimeric polypeptide and IgG1 antibody construct.In some embodiments of any method described herein, IgG1 antibody construct is monoclonal IgG1 antibody, wherein the two antigen-binding domains in monoclonal IgG1 antibody are specifically bound to the joint domain.In some embodiments of any method described herein, IgG1 antibody construct is bispecific IgG1 antibody, wherein one of the two antigen-binding domains in bispecific IgG1 antibody is specifically bound to the joint domain.In some embodiments of any method described herein, contacting step is carried out for a period of time of about 2 hours to about 20 days (e.g., about 1 day to about 15 days).

[0090] In some embodiments of any of the methods described herein, the liquid culture medium is a serum-free liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium is a chemically defined liquid culture medium. In some embodiments of any of the methods described herein, the liquid culture medium comprises serum. In some embodiments of any of the methods described herein, the liquid culture medium comprises the single-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of about 0.5:1 to about 2:1 (e.g., about 0.8:1 to about 1.2:1).

[0091] In some embodiments of any of the methods described herein, the first target binding domain and the linker domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the linker domain and the first domain of a pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the linker domain in the first chimeric polypeptide and the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the second domain of a pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between the second domain of a pair of affinity domains and the second target binding domain in the second chimeric polypeptide.

[0092] In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0093] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises a scFv or a single domain antibody. In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain specifically binds to a target selected from the group consisting of: CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD2 6a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137 , CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BC MA, P-cadherin, CEACAM5, aUL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor and CD28 receptor.

[0094] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine protein, or a 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.

[0095] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some embodiments of any of the methods described herein, the soluble 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, scMHCI, scMHCII, scTCR, a soluble CD155, or a soluble CD28.

[0096] In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains, wherein at least one of the one or more target binding domains is positioned between the linker domain and the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the linker domain and at least one of the one or more target antigen binding domains, and / or a linker sequence between at least one of the one or more target antigen binding domains and the first domain of the pair of affinity domains.

[0097] In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the first target binding domain. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains and the first target binding domain. In some embodiments of any of the methods described herein, at least one of the one or more additional target binding domains is positioned at the N-terminus and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains is positioned between the linker domain in the first chimeric polypeptide and the first domain of the pair of affinity domains. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains positioned at the N-terminus is directly adjacent to the first target binding domain or the first domain of a pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence positioned between the at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of a pair of affinity domains. In some embodiments of any of the methods described herein, in the first chimeric polypeptide, at least one of the one or more additional target binding domains positioned at the C-terminus is directly adjacent to the first target binding domain or the first domain of a pair of affinity domains. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence positioned between the at least one additional target binding domain in the first chimeric polypeptide and the first target binding domain or the first domain of a pair of affinity domains. In some embodiments of any of the methods described herein, at least one of the one or more additional target binding domains positioned between the linker domain and the first domain of a pair of affinity domains is directly adjacent to the linker domain and / or the first domain of the pair of affinity domains.In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence positioned: (i) between the linker domain and at least one of the one or more additional target binding domains, which are positioned between the linker domain and the first domain of the pair of affinity domains, and / or (ii) between the first domain of the pair of affinity domains and at least one of the one or more additional target binding domains, which are positioned between the linker domain and the first domain of the pair of affinity domains.

[0098] In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus and / or C-terminus of the second chimeric polypeptide. In some embodiments of any of the methods described herein, in the second chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the second domain of a pair of affinity domains. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second domain of a pair of affinity domains. In some embodiments of any of the methods described herein, in the second chimeric polypeptide, at least one of the one or more additional target binding domains is directly adjacent to the second target binding domain. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between at least one of the one or more additional target binding domains in the second chimeric polypeptide and the second target binding domain.

[0099] In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments of any of the methods described herein, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to different antigens.

[0100] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains. In some embodiments of any of the methods described herein, the first target binding domain, the second target binding domain, and the one or more other target binding domains are each antigen binding domains. In some embodiments of any of the methods described herein, the antigen binding domain comprises an scFv.

[0101] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains specifically bind to a target selected from the group consisting of CD16a, 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, T NFα, CD26a, CD36, ULBP2, CD30, CD200, CD80, CD86, PD-L2, B7-H4, HVEM, ILT3, ILT4, TIGIT, MHCII, LAG3, CD272, VISTA, CD137, CD40, CD47, CD70, OX40, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, aUL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, Receptor for IL-2, receptor for IL-3, receptor for IL-7, receptor for IL-8, receptor for IL-10, receptor for IL-12, receptor for IL-15, receptor for IL-17, receptor for IL-18, receptor for IL-21, receptor for PDGF-DD, receptor for stem cell factor (SCF), receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), receptor for MICA, receptor for MICB, receptor for ULP16 binding protein, receptor for CD155, receptor for CD122, receptor for CD3, and receptor for CD28.

[0102] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains are soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins. In some embodiments of any of the methods described herein, the soluble interleukins, soluble cytokine proteins, or soluble cell surface proteins are 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.

[0103] In some embodiments of any of the methods described herein, one or more of the first target binding domain, the second target binding domain, and the one or more other target binding domains are soluble interleukin receptors, soluble cytokine receptors, or soluble cell surface receptors. In some embodiments of any of the methods described herein, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.

[0104] In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the first chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a peptide tag at the N-terminus or C-terminus of the second chimeric polypeptide.

[0105] In some embodiments of any of the methods described herein, the linker domain is a soluble tissue factor domain. In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence that is at least 80% identical, at least 90% identical, or at least 95% identical to SEQ ID NO: 1. In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include one or more of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein. In some embodiments of any of the methods described herein, the soluble human tissue factor domain does not include any of the following: a lysine at an amino acid position corresponding to amino acid position 20 of a mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature wild-type human tissue factor protein. In some embodiments of any of the methods described herein, the soluble tissue factor domain is not capable of binding to Factor VIIa. In some embodiments of any of the methods described herein, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments of any of the methods described herein, the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal. In some embodiments of any of the methods described herein, the IgG1 antibody construct comprises at least one antigen binding domain that specifically binds to a soluble tissue factor domain.In some embodiments of any of the methods described herein, the linker domain is selected from the group consisting of a kappa chain and a lambda chain.In some embodiments of any of the methods described herein, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains in the monoclonal IgG1 antibody are specifically bound to a linker domain. In some embodiments of any of the methods described herein, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains in the bispecific IgG1 antibody is specifically bound to a linker domain.

[0106] In some embodiments of any of the methods described herein, the pair of affinity domains is a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. In some embodiments of any of the methods described herein, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments of any of the methods described herein, the human IL15Rα is a mature full-length IL15Rα. In some embodiments of any of the methods described herein, the pair of affinity domains is selected from the group consisting of barnase and barnase inhibitor, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins synaptobrevin, synaptotagmin, synaptophysin, and SNAP25.

[0107] In some embodiments of any of the methods described herein, the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus. In some embodiments of any of the methods described herein, the first chimeric polypeptide and / or the second chimeric polypeptide lacks a signal sequence at its N-terminus.

[0108] In some embodiments of any of the methods described herein, the immune cells are previously obtained from the subject. Some embodiments of any of the methods described herein also include obtaining immune cells from the subject before the contacting step. In some embodiments of any of the methods described herein, the immune cells have previously been genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor. Some embodiments of any of the methods described herein also include introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the immune cells after the contacting step. Some embodiments of any of the methods described herein also include introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the immune cells before the contacting step. Some embodiments of any of the methods described herein also include separating the immune cells after the contacting step. In some embodiments of any of the methods described herein, after the contacting step, the expression level or secretion of one or more proteins selected from the group consisting of TNF-α, IFN-γ, granzyme A, granzyme B, perforin, 2B4, CD8, CD11a, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, C In some embodiments, the method further comprises administering the immune cells to a subject in need thereof after the contacting step.

[0109] In some embodiments of any method described herein, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to age. In some embodiments of any method described herein, the disease or the patient's condition relevant to age is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, loss of lung tissue elasticity relevant to age, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis and renal dysfunction.

[0110] In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing's sarcomas, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-Hodgkin's lymphomas, Hodgkin's lymphomas, 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, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

[0111] In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an infectious disease. In some embodiments of any of the methods described herein, 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, or influenza virus.

[0112] Also provided herein are activated immune cells obtained using any of the methods described herein. Also provided herein are pharmaceutical compositions comprising any of the activated immune cells described herein produced by any of the methods described herein. Also provided herein are kits comprising any of the pharmaceutical compositions described herein, comprising any of the activated immune cells described herein produced by any of the methods described herein.

[0113] Also provided herein are methods of killing cancer cells, infected cells, or senescent cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the activated immune cells described herein produced using any of the methods described herein or any of the pharmaceutical compositions described herein.

[0114] In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing's sarcomas, osteosarcomas, B-cell neoplasms, multiple myeloma, B-cell lymphomas, B-cell non-Hodgkin's lymphomas, Hodgkin's lymphomas, 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, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma.

[0115] In some embodiments of any method described herein, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to aging. In some embodiments of any method described herein, the disease or the patient's condition relevant to aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related 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.

[0116] Also provided herein are methods of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any activated immune cell described herein produced by any of the methods described herein or any pharmaceutical composition described herein. In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having cancer. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of a solid tumor, a hematological tumor, a sarcoma, an osteosarcoma, a glioblastoma, a neuroblastoma, a melanoma, a rhabdomyosarcoma, an Ewing's sarcoma, an osteosarcoma, a B-cell neoplasm, multiple myeloma, a B-cell lymphoma, a B-cell non-Hodgkin's lymphoma, a Hodgkin's lymphoma, a chronic lymphocytic leukemia (CLL), an acute myeloid leukemia (AML), a chronic myeloid leukemia (CML), an acute lymphocytic leukemia (ALL), a myelodysplastic syndrome (MDS), a cutaneous T-cell lymphoma, a retinoblastoma, a gastric cancer, an urothelial cancer, a lung cancer, a renal cell carcinoma, a gastroesophageal cancer, a pancreatic cancer, a prostate cancer, a breast cancer, a colorectal cancer, an ovarian cancer, a non-small cell lung cancer, a head and neck squamous cell carcinoma, an endometrial cancer, a cervical cancer, a liver cancer, and a hepatocellular carcinoma.

[0117] In some embodiments of any method described herein, the subject has been identified or diagnosed as suffering from a disease or the patient's condition relevant to aging. In some embodiments of any method described herein, the disease or the patient's condition relevant to aging is selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatitis fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related 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.

[0118] In some embodiments of any of the methods described herein, the subject has been identified or diagnosed as having an infectious disease. In some embodiments of any of the methods described herein, 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, or influenza virus.

[0119] In some embodiments of any of the methods or kits described herein, the soluble human tissue factor domain does not stimulate blood coagulation. In some embodiments of any of the methods or kits described herein, the soluble tissue factor domain comprises or consists of a sequence from wild-type soluble human tissue factor (or any sequence therefrom).

[0120] Also provided herein is a method for inducing immune cells to differentiate into memory or memory-like immune cells, comprising contacting the immune cells in a liquid culture medium, wherein the liquid culture medium comprises: (1) an effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (a) a first chimeric polypeptide, wherein the first chimeric polypeptide comprises: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains;

[0121] (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the pair of affinity domains; and (2) an effective amount of an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to a linker domain.

[0122] Also provided herein is a method for inducing immune cells to differentiate into memory or memory-like immune cells, comprising contacting the immune cells in a liquid culture medium comprising an effective amount of (i) a single-chain chimeric polypeptide comprising a first target binding domain, a linker domain, and a second target binding domain, and (ii) an IgG1 antibody construct comprising at least one antigen binding domain specifically bound to the linker domain.

[0123] In some embodiments of any of the methods described herein, immune cells are previously obtained from a subject. In some embodiments of any of the methods described herein, immune cells are selected from the group consisting of immature thymocytes, peripheral blood Treg cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, λδT cells, αβT 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 of any of the methods described herein, immune cells have previously been genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor.

[0124] Some embodiments of any of the methods described herein further comprise introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the immune cell after the contacting step. Some embodiments of any of the methods described herein further comprise administering the immune cell to a subject in need thereof.

[0125] As used herein, the term "chimeric" refers to a polypeptide that includes amino acid sequences (e.g., domains) that were originally derived from two different sources (e.g., two different naturally occurring proteins, e.g., from the same or different species). For example, a chimeric polypeptide can include domains from at least two different naturally occurring human proteins. In some instances, 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).

[0126] An "antigen binding domain" is one or more protein domains (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 antigens. In some examples, the antigen binding domain may bind to an antigen or epitope with a specificity and affinity similar to that of 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 include an alternative scaffold. Non-limiting examples of antigen binding domains are described herein. Other examples of antigen binding domains are known in the art.

[0127] A "soluble tissue factor domain" refers to a polypeptide 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 a segment of a wild-type mammalian tissue factor protein (e.g., a wild-type human tissue factor protein) that lacks the transmembrane domain and the intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

[0128] The term "soluble interleukin protein" is used herein to refer to a mature and secreted interleukin protein or a biologically active fragment thereof. In some instances, the soluble interleukin protein can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature and secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein), and retains its biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

[0129] The term "soluble cytokine protein" is used herein to refer to a mature and secreted cytokine protein or a biologically active fragment thereof. In some instances, the soluble cytokine protein can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature and secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein), and retains its biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

[0130] The term "soluble interleukin receptor" is used herein in the broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) capable of binding to one or more of its natural ligands (e.g., under physiological conditions, such as at room temperature, in phosphate-buffered saline). 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 its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally, also lacks its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0131] The term "soluble cytokine receptor" is used herein in the broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) capable of binding one or more of its natural ligands (e.g., under physiological conditions, such as at room temperature, in phosphate-buffered saline). For example, a soluble cytokine receptor 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 the extracellular domain of a wild-type cytokine receptor and retains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally, also lacks its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0132] The term "ligand of a co-stimulatory molecule" is used herein in the broadest sense to refer to a polypeptide that is capable of binding to and activating a co-stimulatory receptor molecule on an immune cell (e.g., under physiological conditions, such as at room temperature, in phosphate-buffered saline). For example, the ligand of a co-stimulatory molecule can include a sequence that is at least 70% identical to the ligand of a co-stimulatory molecule (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) that retains its ability to specifically bind to one or more of its natural receptors. Non-limiting examples of ligands of co-stimulatory molecules are described herein.

[0133] The term "antibody" is used herein in the broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, complete antibodies (e.g., complete immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. Other examples of antibodies are described herein. Other examples of antibodies are known in the art.

[0134] "Affinity" refers to the strength of the sum of the non-covalent interactions between an antigen binding site and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between a member of an antigen binding domain and an antigen or epitope. The affinity of a molecule X for its partner Y can be measured by the dissociation equilibrium constant (K D ) is represented. 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 the methods described herein. Affinity can be measured, for example, using surface plasmon resonance (SPR) technology (e.g., ) or biolayer interferometry (e.g., Other methods for determining affinity for an antigen binding domain and its corresponding antigen or epitope are known in the art.

[0135] As used herein, "single-chain polypeptide" refers to a single protein chain.

[0136] The term "a pair of affinity domains" is based on a number of factors less than 1 × 10 -7 M (e.g., less than 1×10 -8 M, less than 1×10 - 9 M, less than 1×10 -10 M or less than 1×10 -11 M)K DTwo different protein domains that specifically bind to each other. In some instances, a pair of affinity domains can be a pair of naturally occurring proteins. In some embodiments, a pair of affinity domains can be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs are described herein.

[0137] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen binding domain. An epitope can, for example, be composed of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former, but not to the latter, may be lost in the presence of denaturing solvents. An epitope can include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. Methods for identifying epitopes that bind to antigen binding domains are known in the art.

[0138] "Immune effector cell" refers to a cell of the immune system of a mammal that can directly or indirectly identify pathogenic cells (e.g., cancer cells) in a mammal and / or cause cell arrest or cell death of the cell. Non-limiting examples of immune effector cells include macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes and T helper cells), neutrophils, monocytes, and eosinophils. Other examples of immune effector cells are known in the art.

[0139] The term "treating" refers to ameliorating at least one symptom of a condition. In some instances, the condition being treated is cancer, and ameliorating at least one symptom of cancer includes reducing abnormal proliferation, gene expression, signaling, translation, and / or secretion of a factor. Generally, a treatment method comprises administering a therapeutically effective amount of a composition to a subject in need of or identified as in need of such treatment, wherein the composition reduces at least one symptom of the condition.

[0140] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Methods and materials for use with the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and 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 a conflict, the present specification (including definitions) shall prevail.

[0141] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1A schematic diagram showing an exemplary IL-7 / IL-15RαSu DNA construct.

[0143] Figure 2 A schematic diagram showing an exemplary IL-21 / / TF / IL-15 DNA construct.

[0144] Figure 3 Schematic diagram showing the interaction between an exemplary IL-7 / IL-15RαSu and IL-21 / TF / IL-15 DNA construct.

[0145] Figure 4 Schematic diagram showing the interaction between an exemplary IL-7 / IL-15RαSu and IL-21 / TF / IL-15 fusion protein to produce the IL-21 / TF / IL-15:IL-7 / IL-15RαSu complex (21t15-7s).

[0146] Figure 5 Shown is the expansion of primary natural killer (NK) cells by stimulation with 21t15-7s + anti-TF IgG1 antibody.

[0147] Figure 6 Activation of expanded primary NK cells is shown using CD25 MFI and CD69 MFI as markers of NK cell activation.

[0148] Figure 7 The cytotoxic activity of the expanded NK cells against K562 human tumor cells was shown, wherein NK cells stimulated with 21t15-7s+anti-TF IgG1 antibody exhibited stronger K562 cell-specific lysis than NK cells not stimulated with 21t15-7s+anti-TF IgG1 antibody.

[0149] Figure 8 A schematic diagram showing an exemplary TGF-βRII / IL-15RαSu DNA construct.

[0150] Figure 9 A schematic diagram showing an exemplary IL-21 / TF / IL-15 DNA construct.

[0151] Figure 10 Schematic diagram showing the interaction between an exemplary TGF-βRII / IL-15RαSu and IL-21 / TF / IL-15 DNA construct.

[0152] Figure 11Schematic diagram showing the interaction between an exemplary TGF-βRII / IL-15RαSu and IL-21 / TF / IL-15 fusion protein to produce the IL-21 / TF / IL-15:TGF-βRII / IL-15RαSU complex (21t15-TGFRs).

[0153] Figure 12 Shown is the expansion of primary natural killer (NK) cells stimulated with 21t15-TGFRs + anti-TF IgG1 antibody (dark line with squares) or contacted with 21t15-TGFRs alone, anti-TF IgG1 antibody, anti-TF IgG4 antibody, or the combination of 21t15-TGFRs + anti-TF IgG4 antibody (all other data shown).

[0154] Figure 13 Shown are the activation of expanded primary NK cells using CD25 MFI (top) and CD69 MFI (bottom) as markers of NK cell activation.

[0155] Figure 14 A schematic diagram showing an exemplary IL-21 / IL-15RαSu DNA construct.

[0156] Figure 15 A schematic diagram showing an exemplary IL-7 / TF / IL-15 DNA construct.

[0157] Figure 16 Schematic diagram showing the interaction between an exemplary IL-21 / IL-15RαSu and IL-7 / TF / IL-15 DNA construct.

[0158] Figure 17 Schematic diagram showing the interaction between an exemplary IL-21 / IL-15RαSu and IL-7 / TF / IL-15 fusion protein to produce the IL-7 / TF / IL-15:IL-21 / IL-15RαSu complex (7t15-21s).

[0159] Figure 18 Schematic representation of the activation and expansion of primary natural killer (NK) cells by stimulation with 21t15-TGFRs + anti-TF IgG1 antibody.

[0160] Figure 19 Shown are size exclusion chromatography (SEC) profiles of anti-TF IgG1 antibody, 7t15-21s, and a complex containing equal amounts of anti-TF IgG1 antibody and 7t15-21s.

[0161] Figure 20is a graph showing the oxygen consumption rate (OCR) of human NK cells (in picomoles / minute (pmol / min)) isolated from blood (1×10 6 Cells were maintained at 0.5 × 10 cells / ml and stimulated with (1) 50 nM anti-TF IgG1 (IgG1), (2) 100 nM 7t15-21s and 50 nM anti-TF IgG1, or (3) 100 nM 21t15-7s and 50 nM anti-TF IgG1 for up to 5 days in RPMI 1640 supplemented with 4 mM L-glutamine, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, and 10% fetal bovine serum at 37°C, 5% CO2. 6 cells / ml to 2.0×10 6 Cells were cultured at a concentration of 10 cells / mL until day 5. Glycolytic stress assays were performed in Seahorse medium containing 2 mM glutamine. Drug concentrations used during the assay were as follows: 10 mM glucose, 100 nM oligomycin, and 100 mM 2-deoxy-D-glucose.

[0162] Figure 21 is a graph showing the extracellular acidification rate (ECAR) (in mPH / min) of human NK cells isolated from blood (1×10 6 Cells were maintained at 0.5 × 10 cells / ml and stimulated with (1) 50 nM anti-TF IgG1 (IgG1), (2) 100 nM 21t15-7s and 50 nM anti-TF IgG1, or (3) 100 nM 21t15-7s and 50 nM anti-TF IgG1 for up to 5 days in RPMI 1640 supplemented with 4 mM L-glutamine, penicillin, streptomycin, nonessential amino acids, sodium pyruvate, and 10% fetal bovine serum at 37°C, 5% CO2. 6 cells / ml to 2.0×10 6 Cells were cultured at a concentration of 10 cells / mL until day 5. Glycolytic stress assays were performed in Seahorse medium containing 2 mM glutamine. Drug concentrations used during the assay were as follows: 10 mM glucose, 100 nM oligomycin, and 100 mM 2-deoxy-D-glucose.

[0163] Figure 22 is a schematic diagram showing the structure of the 18t15-12s construct.

[0164] Figure 23 Figure 2 shows human NK cells (2×10 6Two graphs of oxygen consumption rate (OCR) (in pmol / min) of cells / ml) were plotted. The isolated NK cells were kept unstimulated or stimulated overnight with (1) 100 nM 18t15-12s ("1812") or (2) a mixture of recombinant human IL-12 (0.25 μg), recombinant human IL-15 (1.25 μg) and recombinant human IL-18 (1.25 μg) ("single cytokine") in RPMI 1640 supplemented with 4 mM L-glutamine, penicillin, streptomycin, non-essential amino acids, sodium pyruvate and 10% fetal bovine serum at 37°C, 5% CO2. The next day, the cells were harvested and the expanded NK cells were subjected to extracellular flux analysis using an XFp analyzer (Seahorse Bioscience). The harvested cells were washed and plated at least twice at 2.0 × 10 5 Cells were plated per well for extracellular flux analysis of oxygen consumption rate. Glycolytic stress assays were performed in Seahorse medium containing 2 mM glutamine. Drug concentrations used during the assay were: 10 mM glucose, 100 nM oligomycin, and 100 mM 2-deoxy-D-glucose.

[0165] Figure 24 The results show that human NK cells (2×10 6 Two graphs of the extracellular acidification rate (ECAR) (in mPH / min) of 1812 cells / ml) were plotted. The isolated NK cells were kept unstimulated or stimulated overnight with (1) 100 nM 18t15-12s ("1812") or (2) a mixture of recombinant human IL-12 (0.25 μg), recombinant human IL-15 (1.25 μg) and recombinant human IL-18 (1.25 μg) ("single cytokine") in RPMI 1640 supplemented with 4 mM L-glutamine, penicillin, streptomycin, non-essential amino acids, sodium pyruvate and 10% fetal bovine serum at 37°C, 5% CO2. The next day, the cells were harvested and the expanded NK cells were subjected to extracellular flux analysis using an XFp analyzer (Seahorse Bioscience). The harvested cells were washed and plated at least twice at 2.0 × 10 6 Cells were plated per well for extracellular flux analysis (ECAR). Glycolytic stress testing was performed in Seahorse medium containing 2 mM glutamine. Drug concentrations during the assay were: 10 mM glucose, 100 nM oligomycin, and 100 mM 2-deoxy-D-glucose.

[0166] Figure 25 A schematic diagram showing an exemplary IL-12 / IL-15RαSu DNA construct.

[0167] Figure 26 A schematic diagram showing an exemplary IL-18 / TF / IL-15 DNA construct.

[0168] Figure 27 Schematic diagram showing the interaction between an exemplary IL-12 / IL-15RαSu and IL-18 / TF / IL-15 DNA construct.

[0169] Figure 28 Schematic diagram showing the interaction between an exemplary IL-12 / IL-15RαSu and IL-18 / TF / IL-15 fusion protein to produce the IL-18 / TF / IL-15:IL-12 / IL-15RαSu complex (18t15-12s).

[0170] Figure 29 Chromatogram showing the elution of 18t15-12s purification from an anti-TF affinity column.

[0171] Figure 30 Shown is an exemplary chromatogram of anti-TF antibody / SEC purified 18t15-12s protein after elution on an analytical size exclusion column, demonstrating separation of monomeric multiprotein 18t15-12s complex from protein aggregates.

[0172] Figure 31 An example of 4-12% SDS-PAGE showing the 18t15-12s complex after disulfide bond reduction: Lane 1: SeeBlue Plus2 marker; Lane 2: 18t15-12s purified with anti-TF antibody (0.5 μg); Lane 3: 18t15-12s purified with anti-TF antibody (1 μg).

[0173] Figure 32 SDS PAGE analysis of deglycosylated and non-deglycosylated 18t15-12s is shown. Lane 1: 18t15-12s (0.5 μg) purified with anti-TF antibody, non-deglycosylated; Lane 2: 18t15-12s (1 μg) purified with anti-TF antibody, non-deglycosylated; Lane 3: 18t15-12s (1 μg), deglycosylated; Lane 4: Manufacturer's Mark12 unstained.

[0174] Figure 33 Shown is a sandwich ELISA for the 18t15-12s complex comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 detection antibody (BAF 219).

[0175] Figure 34 Shown is a sandwich ELISA for the 18t15-12s complex comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247).

[0176] Figure 35 Shown is a sandwich ELISA for the 18t15-12s complex comprising an anti-human tissue factor capture antibody and a biotinylated anti-human IL-18 detection antibody (D045-6).

[0177] Figure 36 Shown is a sandwich ELISA for the 18t15-12s complex containing anti-human tissue factor (I43) capture antibody and anti-human tissue factor detection antibody.

[0178] Figure 37 Shown are IL-15-dependent 32Dβ cell proliferation mediated by the 18t15-12s complex (open squares) and recombinant IL-15 (black squares).

[0179] Figure 38 The bioactivity of IL-18 within the 18t15-12s complex (open squares) is shown, where recombinant IL-18 (black squares) and recombinant IL-12 (black circles) were used as positive and negative controls, respectively.

[0180] Figure 39 The bioactivity of IL-12 within the 18t15-12s complex (open squares) is shown, where recombinant IL-12 (black circles) and recombinant IL-18 (open squares) were used as positive and negative controls, respectively.

[0181] Figure 40A Flow cytometry graphs showing cell surface CD25 expression on NK cells induced by the 18T15-12S complex. Figure 40B Flow cytometry graphs showing cell surface CD69 expression on NK cells induced by the 18T15-12S complex.

[0182] Figure 41 Flow cytometry graphs showing intracellular interferon-γ expression in NK cells induced by the 18T15-12S complex.

[0183] Figure 42 A bar graph is shown, which shows the cytotoxicity of human NK cells induced by 18T15-12S against K562. 1×10 5 K562 cells (labeled with Celltrace Violet) and 2×10 5 Human NK cells (NK cells were isolated from human blood buffy coat using the StemCell Human NK Cell Purification Kit) were cultured with 18t15-12s in RPMI-10 for 20 hours. The cell mixture was harvested, stained with propidium iodide (PI), and analyzed using BD FACS Celesta. TMFlow cytometric analysis. The graphs represent replicate samples.

[0184] Figure 43 A schematic diagram showing an exemplary IL-12 / IL-15RαSu / αCD16 DNA construct.

[0185] Figure 44 A schematic diagram showing an exemplary IL-18 / TF / IL-15 DNA construct.

[0186] Figure 45 Schematic diagram showing the interaction between an exemplary IL-12 / IL-15RαSu / αCD16 scFv and an IL-18 / TF / IL-15 DNA construct.

[0187] Figure 46 Schematic diagram showing an exemplary 18t15-12s / αCD16 protein complex.

[0188] Figure 47 Shown are sandwich ELISAs for the 18t15-12s16 complex containing anti-human tissue factor capture antibody and biotinylated anti-human IL-12 (dark lines) or anti-human tissue factor detection antibodies (light lines).

[0189] Figure 48 A schematic diagram showing an exemplary TGF-βRII / IL-15RαSu DNA construct.

[0190] Figure 49 A schematic diagram showing an exemplary IL-21 / TF / IL-15 construct.

[0191] Figure 50 Schematic diagram showing the interaction between exemplary IL-IL-21 / TF / IL-15 and TGFβRII / IL-15RαSu constructs.

[0192] Figure 51 Schematic diagram showing the interaction between an exemplary TGFβRII / IL-15RαSu and IL-21 / TF / IL-15 fusion protein to produce the IL-21 / TF / IL-15 / TGFβRII / IL-15RαSu complex (21t15-TGFRs).

[0193] Figure 52 A chromatogram showing the purification elution of 21t15-TGFRs from an anti-TF antibody affinity column.

[0194] Figure 53 An exemplary size exclusion chromatogram of 21t15-TGFRs is shown, showing a major protein peak and a high molecular weight peak.

[0195] Figure 54 An example of a 4-12% SDS-PAGE of the 21t15-TGFRs complex after disulfide bond reduction is shown. Lane 1: Mark 12 unstained marker (numbers on the left indicate molecular weight in kDa); Lane 2: 21t15-TGFRs (0.5 μg); Lane 3: 21t15-TGFRs (1 μg); Lane 4: 21t15-TGFRs, deglycosylated (1 μg), where the MW is the expected size of 53 kDa and 39.08 kDa.

[0196] Figure 55 Shown is a sandwich ELISA for the 21t15-TGFRs complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-21 detection antibody (13-7218-81, BioLegend).

[0197] Figure 56 Shown is a sandwich ELISA for the 21t15-TGFRs complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247, R&D Systems).

[0198] Figure 57 Shown is a sandwich ELISA for the 21t15-TGFRs complex containing anti-human tissue factor capture antibody and biotinylated anti-human TGFβRII detection antibody (BAF241, R&D Systems).

[0199] Figure 58 Shown is a sandwich ELISA for the 21t15-TGFRs complex containing anti-human tissue factor (I43) capture antibody and anti-human tissue factor detection antibody.

[0200] Figure 59 Shown are IL-15-dependent 32Dβ cell proliferation mediated by the 21t15-TGFRs complex (open squares) compared to IL-15 (black squares).

[0201] Figure 60 The biological activity of the TGFβRII domain within the 21t15-TGFRs complex (open squares) is shown. TGFβRII / Fc (black squares) served as a positive control.

[0202] Figure 61A Flow cytometry graphs showing cell surface CD25 expression on NK cells induced by the 21t15-TGFRs complex. Figure 61B Flow cytometry graphs showing cell surface CD69 expression on NK cells induced by the 21t15-TGFRs complex.

[0203] Figure 62 Flow cytometry graphs showing intracellular interferon-γ expression in NK cells induced by the 21t15-TGFRs complex.

[0204] Figure 63 A bar graph is shown, which shows the cytotoxicity of human NK cells against K562 induced by 21t15-TGFRs. 1×10 5 K562 cells (labeled with Celltrace Violet) and 2×10 5 Human NK cells (NK cells were isolated from human blood buffy coat using the StemCell Human NK Cell Purification Kit) were cultured with 18t15-12s in RPMI-10 for 20 hours. The cell mixture was harvested, stained with propidium iodide (PI), and analyzed using a BD FACSCelestat. TM Flow cytometric analysis. The graphs represent replicate samples.

[0205] Figure 64 is a schematic diagram of an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide.

[0206] Figure 65 is a chromatogram showing the elution of an exemplary αCD3scFv / TF / αCD28scFv single chain chimeric polypeptide from an anti-tissue factor affinity column.

[0207] Figure 66 is a chromatogram showing the elution of a Superdex200 Increase 10 / 300GL gel filtration column loaded with an exemplary αCD3scFv / TF / αCD28scFv single chain chimeric polypeptide.

[0208] Figure 67 A sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage Bis-Tris gel) of an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide purified using an anti-tissue factor affinity column.

[0209] Figure 68 is a graph showing ELISA quantification of an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide using the method described in Example 53. Purified tissue factor was used as a control.

[0210] Figure 69 The present invention shows that the exemplary αCD3scFv / TF / αCD28scFv single chain chimeric polypeptide stimulates CD4 isolated from the blood of two donors. + Graph of the ability of T cells to express CD25. The experiments were performed as described in Example 54.

[0211] Figure 70 The present invention shows that the exemplary αCD3scFv / TF / αCD28scFv single chain chimeric polypeptide stimulates CD8 isolated from the blood of two donors. + Graph of the ability of T cells to express CD25. The experiments were performed as described in Example 54.

[0212] Figure 71 The present invention shows that the exemplary αCD3scFv / TF / αCD28scFv single chain chimeric polypeptide stimulates CD4 isolated from the blood of two donors. + Figure 5. Graph of the ability of T cells to express CD69. The experiment was performed as described in Example 54.

[0213] Figure 72 A schematic diagram showing an exemplary IL-7 / IL-15RαSu DNA construct.

[0214] Figure 73 A schematic diagram showing an exemplary IL-21 / TF / IL-15 DNA construct.

[0215] Figure 74 Schematic diagram showing the interaction between an exemplary IL-7 / IL-15RαSu and IL-21 / TF / IL-15 DNA construct.

[0216] Figure 75 Schematic diagram showing the interaction between an exemplary IL-7 / IL-15RαSu and IL-21 / TF / IL-15 fusion protein to produce the IL-21 / TF / IL-15:IL-7 / IL-15RαSu complex (21t15-7s).

[0217] Figure 76 A schematic diagram showing an exemplary TGF-βRII / IL-15RαSu DNA construct.

[0218] Figure 77 A schematic diagram showing an exemplary IL-21 / TF / IL-15 DNA construct.

[0219] Figure 78 Schematic diagram showing the interaction between an exemplary TGF-βRII / IL-15RαSu and IL-21 / TF / IL-15 DNA construct.

[0220] Figure 79 Schematic diagram showing the interaction between an exemplary TGF-βRII / IL-15RαSu and IL-21 / TF / IL-15 fusion protein to produce the IL-21 / TF / IL-15:TGF-βRII / IL-15RαSU complex (21t15-TGFR).

[0221] Figure 80 A schematic diagram showing an exemplary IL-21 / IL-15RαSu DNA construct.

[0222] Figure 81 A schematic diagram showing an exemplary IL-7 / TF / IL-15 DNA construct.

[0223] Figure 82 Schematic diagram showing the interaction between an exemplary IL-21 / IL-15RαSu and IL-7 / TF / IL-15 DNA construct.

[0224] Figure 83 Schematic diagram showing the interaction between an exemplary IL-21 / IL-15RαSu and IL-7 / TF / IL-15 fusion protein to produce the IL-7 / TF / IL-15:IL-21 / IL-15RαSU complex (7t15-21s).

[0225] Figure 84 Shown are size exclusion chromatography (SEC) profiles of anti-TF IgG1 antibody, 7t15-21s, and a complex containing equal amounts of anti-TF IgG1 antibody and 7t15-21s.

[0226] Figure 85 Schematic diagram showing the 7t15-16s21 construct.

[0227] Figure 86 Another schematic diagram showing the 7t15-16s21 construct.

[0228] Figure 87A and 87B is a graph showing the binding of 7t15-16s21 to CHO cells expressing human CD16b, compared to a control protein.

[0229] Figures 88A-88C Results from an ELISA assay detecting 7t15-16s21 using antibodies against IL-15, IL-21, and IL-7.

[0230] Figure 89 Shown are the results of 32Dβ cell proliferation assays using 7t15-16s21 or recombinant IL-15.

[0231] Figure 90 is a line graph showing the chromatogram of cell culture supernatant containing 7t15-16s21 protein after binding and elution on anti-TF antibody resin.

[0232] Figure 91 Analytical SEC profile of 7t15-16s21 is shown.

[0233] Figure 92 Schematic diagram showing the TGFRt15-16s21 construct.

[0234] Figure 93 Another schematic diagram showing the TGFRt15-16s21 construct.

[0235] Figure 94A and 94B The binding affinity of TGFRt15-16s21 and 7t15-21s to CHO cells expressing human CD16b is shown. Figure 94A The binding affinity of TGFRt15-16s21 to CHO cells expressing human CD16b is shown. Figure 94B The binding affinity of 7t15-21s to CHO cells expressing human CD16b is shown.

[0236] Figure 95 Shown are the results of TGFβ1 inhibition by TGFRt15-16s21 and TGFR-Fc.

[0237] Figure 96 Shown are the results of 32Dβ cell proliferation analysis using TGFRt15-16s21 or recombinant IL-15.

[0238] Figures 97A-97C The results of detecting IL-15, IL-21, and TGFβRII in TGFRt15-16s21 using ELISA with corresponding antibodies are shown.

[0239] Figure 98 It is a line graph showing the chromatogram of cell culture supernatant containing TGFRt15-16s21 protein after binding and elution on anti-TF antibody resin.

[0240] Figure 99 Shown are the results of reducing SDS-PAGE analysis of TGFRt15-16s21.

[0241] Figure 100 Schematic diagram showing the 7t15-7s construct.

[0242] Figure 101 Another schematic diagram showing the 7t15-7s construct.

[0243] Figure 102 is a line graph showing the chromatogram of cell culture supernatant containing 7t15-7s protein after binding and elution on anti-TF antibody resin.

[0244] Figure 103Detection of TF, IL-15, and IL-7 in 7t15-7s using ELISA is shown.

[0245] Figure 104A and 104B Shown are spleen weights and percentages of immune cell types from mice treated with 7t15-7s and controls. Figure 115A Shown are spleen weights of mice treated with 7t15-7s compared to PBS controls. Figure 115B showed that CD4 + T cells, CD8 + The percentages of T cells and NK cells.

[0246] Figure 105 Schematic diagram showing the TGFRt15-TGFRs construct.

[0247] Figure 106 Another schematic diagram showing the TGFRt15-TGFRs construct.

[0248] Figure 107 Shown are the results of TGFβ1 inhibition by TGFRt15-TGFRs and TGFR-Fc.

[0249] Figure 108 Shown are the results of 32Dβ cell proliferation assays using TGFRt15-TGFRs or recombinant IL-15.

[0250] Figure 109A and 109B The results of detecting IL-15 and TGFβRII in TGFRt15-TGFRs using ELISA with corresponding antibodies are shown.

[0251] Figure 110 It is a line graph showing the chromatogram of cell culture supernatant containing TGFRt15-TGFRs protein after binding and elution on anti-TF antibody resin.

[0252] Figure 111 Analytical SEC profile of TGFRt15-TGFRs is shown.

[0253] Figure 112 Shown are TGFRt15-TGFR before and after deglycosylation as analyzed by reducing SDS-PAGE.

[0254] Figure 113A and 113B Shown are the spleen weights and percentages of immune cell types of mice treated with TGFRt15-TGFRs and controls. Figure 113AShown are the spleen weights of mice treated with TGFRt15-TGFRs compared with PBS controls. Figure 113B showed that CD4 + T cells, CD8 + The percentages of T cells and NK cells.

[0255] Figure 114A and 114B Shown are spleen weights and immune stimulation over 92 hours in mice treated with TGFRt15-TGFRs. Figure 114A Shown are the spleen weights of mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figure 114B Shown are the percentages of immune cells in mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment.

[0256] Figure 115A and 115B Shown are the expressions of Ki67 and granzyme B in mice treated with TGFRt15-TGFRs over time.

[0257] Figure 116 showed that TGFRt15-TGFRs enhanced the cytotoxicity of splenocytes in C57BL / 6 mice.

[0258] Figure 117 Shown are changes in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or chemotherapy in combination with TGFRt15-TGFRs in a pancreatic cancer mouse model.

[0259] Figure 118 The cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFRs is shown.

[0260] Figures 119A-119E Results showing the in vivo efficacy of TGFRt15-TGFRs in a melanoma mouse model. Figure 119A Schematic diagram showing the treatment scheme. Figure 119B Shown are tumor volumes over time following saline treatment, chemotherapy (DTX), or a combination of chemotherapy (DTX), TGFRt15-TGFRs, and TA99 (anti-TRP1 antibody). Figure 119C Peripheral blood analysis using fluorescently labeled antibodies against NK1.1 is shown. Figure 119D Peripheral blood analysis using fluorescently labeled antibodies against CD8 is shown. Figure 119E Peripheral blood analysis using fluorescently labeled antibodies against CD4 is shown.

[0261] Figure 120 Schematic diagram showing the 7t15-21s137L (long version) construct.

[0262] Figure 121 Another schematic diagram showing the 7t15-21s137L (long version) construct.

[0263] Figure 122 is a line graph showing the chromatogram of cell culture supernatant containing 7t15-21s137L (long version) protein after binding and elution on anti-TF antibody resin.

[0264] Figure 123 Analytical SEC profile of 7t15-21s137L (long version) is shown.

[0265] Figure 124 Binding of 7t15-21s137L (short version) to CD137L (4.1BBL) is shown.

[0266] Figures 125A-125C Detection of IL15, IL21 and IL7 in 7t15-21s137L (short version) by ELISA is shown. Figure 125A Detection of IL15 in 7t15-21s137L (short version) by ELISA is shown. Figure 125B Detection of IL21 in 7t15-21s137L (short version) by ELISA is shown. Figure 125C Detection of IL7 in 7t15-21s137L (short version) by ELISA is shown.

[0267] Figure 126 Results from CTLL-2 cell proliferation analysis are shown.

[0268] Figure 127 Showing the activity of 7t15-1s137L (short version) in promoting the proliferation of IL21R-containing B9 cells.

[0269] Figure 128 Schematic diagram showing the 7t15-TGFRs construct.

[0270] Figure 129 Another schematic diagram showing the 7t15-TGFRs construct.

[0271] Figure 130 Shown are the results of TGFβ1 inhibition by 7t15-TGFR and TGFR-Fc.

[0272] Figures 131A-131CDetection of IL-15, TGFβRII, and IL-7 in 7t15-TGFRs by ELISA is shown.

[0273] Figure 132 Shown are the results of 32Dβ cell proliferation assays using 7t15-TGFRs or recombinant IL-15.

[0274] Figure 133 It is a line graph showing the chromatogram of cell culture supernatant containing 7t15-TGFRs protein after binding and elution on anti-TF antibody resin.

[0275] Figure 134 Shown are 7t15-TGFRs before and after deglycosylation as analyzed using reducing SDS-PAGE.

[0276] Figure 135 ELISA detection of IL-7, IL-15 and TGFβRII in 7t15-TGFRs protein is shown.

[0277] Figure 136A and 136B Shown are the spleen weights and percentages of immune cell types of mice treated with 7t15-TGFRs and controls. Figure 136A Shown are the spleen weights of mice treated with 7t15-TGFRs compared to PBS controls. Figure 136B showed that compared with PBS control, CD4 + T cells, CD8 + The percentages of T cells and NK cells.

[0278] Figure 137A and 137B showed that 7t15-TGFRs induced CD4 + and CD8 + Upregulation of CD44 expression on T cells.

[0279] Figure 138A and 138B showed that 7t15-TGFRs can regulate CD8 + Upregulation of Ki67 expression and granzyme B expression in T cells and NK cells.

[0280] Figure 139 showed that 7t15-TGFRs enhanced the cytotoxicity of splenocytes in C57BL / 6 mice.

[0281] Figure 140 Schematic diagram showing the TGFRt15-21s137L construct.

[0282] Figure 141 Another schematic diagram showing the TGFRt15-21s137L construct.

[0283] Figure 142 It is a line graph showing the chromatogram of cell culture supernatant containing TGFRt15-21s137L protein after binding and elution on anti-TF antibody resin.

[0284] Figure 143 Schematic diagram showing the TGFRt15-TGFRs21 construct.

[0285] Figure 144 Another schematic diagram showing the TGFRt15-TGFRs21 construct.

[0286] Figure 145 is a line graph showing the chromatogram of cell culture supernatant containing TGFRt15-TGFRs21 protein after binding and elution on anti-TF antibody resin.

[0287] Figure 146 Shown are TGFRt15-TGFRs21 before and after deglycosylation analysis by reducing SDS-PAGE.

[0288] Figure 147A and 147B Detection of TGFRt15-TGFRs21 components using ELISA is shown.

[0289] Figure 148A and 148B Showing the presence of CD4 in the spleens of control-treated and TGFRt15-TGFRs21-treated mice + T cells, CD8 + Percentage and proliferation of T cells and natural killer (NK) cells.

[0290] Figure 149 showed upregulation of granzyme B expression by splenocytes in mice treated with TGFRt15-TGFRs21.

[0291] Figure 150 showed that TGFRt15-TGFRs21 enhanced the cytotoxicity of splenocytes in C57BL / 6 mice.

[0292] Figure 151 Schematic diagram showing the TGFRt15-TGFRs16 construct.

[0293] Figure 152 Another schematic diagram showing the TGFRt15-TGFRs16 construct.

[0294] Figure 153 Schematic diagram showing the TGFRt15-TGFRs137L construct.

[0295] Figure 154 Another schematic diagram showing the TGFRt15-TGFRs137L construct.

[0296] Figure 155 is a schematic diagram of an exemplary 2t2 single-chain chimeric polypeptide.

[0297] Figure 156 Shown is IL-2 activity in 2t2 compared to recombinant IL-2 measured using a 32D beta cell proliferation assay.

[0298] Figure 157 Shown is IL-2 activity in 2t2 compared to recombinant IL-2 measured using a CTLL-2 cell proliferation assay.

[0299] Figure 158 ApoE cells fed with standard chow or high-fat diet and treated with PBS control (untreated) or 2t2 - / - Fasting blood glucose levels in mice.

[0300] Figure 159 ApoE cells fed with standard chow or high-fat diet and treated with PBS control (untreated) or 2t2 - / - CD4 in mouse blood lymphocytes + CD25 + FoxP3 + The ratio of T regulatory cells.

[0301] Figure 160 is a line graph showing the chromatogram of cell culture supernatant containing 2t2 protein after binding and elution on anti-TF antibody resin.

[0302] Figure 161 Analytical SEC profile of 2t2 is shown.

[0303] Figure 162A and 162B Shown is the reducing SDS-PAGE analysis of 2t2 before and after deglycosylation. Figure 162A Shown is reducing SDS-PAGE analysis of 2t2 before deglycosylation. Figure 162B Shown is reducing SDS-PAGE analysis of 2t2 after deglycosylation.

[0304] Figure 163A and 163B Shown are the results of immunostimulation of C57BL / 6 mice using 2t2. Figure 163A Shown are spleen weights after treatment with 2t2. Figure 163BShown are the percentages of immune cell types after treatment with 2T2.

[0305] Figure 164 showed that CD4 + CD25 expression is upregulated on T cells.

[0306] Figure 165 The pharmacokinetics of 2t2 in C57BL / 6 mice are shown.

[0307] Figure 166A and 166B Showing that 2t2 can alleviate ApoE - / - Effects of high fat diet on atherosclerotic plaque formation in mice. Figure 166A ApoE-positive mice fed a standard chow or high-fat diet and treated with PBS control or 2t2 - / - Representative views of atherosclerotic plaques in mice. Figure 166B The quantitative analysis results of atherosclerotic plaques in each group are shown.

[0308] Figure 167 Shown are fasting blood glucose levels in mice treated with 2t2 compared to mice treated with controls.

[0309] Figure 168 CD4 in blood lymphocytes from mice treated with 2t2 and mice treated with control + CD25 + FoxP3 + The percentage of Tregs.

[0310] Figure 169 is a schematic diagram of an exemplary 15t15 single-chain chimeric polypeptide.

[0311] Figure 170 Shown is the IL-15 activity of 15t15 compared to recombinant IL-15 in a 32Dβ cell proliferation assay.

[0312] Figure 171 is a line graph showing the chromatogram of cell culture supernatant containing 15t15 protein after binding and elution on anti-TF resin.

[0313] Figure 172A and 172B Shown is the reducing SDS-PAGE analysis of 15t15 before and after deglycosylation. Figure 172A Shown is reducing SDS-PAGE analysis of 15t15 before deglycosylation. Figure 172B Shown is reducing SDS-PAGE analysis of 15t15 after deglycosylation.

[0314] Figure 173is a set of histograms and a set of graphs showing the changes in the surface phenotype of NK cells after stimulation with 18t15-12s, 18t15-12s16, and 7t15-21s+anti-TF antibody.

[0315] Figure 174 is a set of graphs showing changes in the surface phenotype of lymphocyte populations after stimulation with: 18t15-12s; 18t15-12s16; a mixture of single cytokines, rhIL15, rhIL18, and rhIL-12; 7t15-21s+anti-TF antibody; 7t15-21s; or anti-TF antibody.

[0316] Figure 175 is a graph showing the increased proliferation of NK cells in vitro after stimulation with the multi-chain construct alone or in the presence of anti-TF IgG1 or anti-TF IgG4.

[0317] Figure 176 is a graph showing increased in vitro proliferation of NK cells over time following stimulation with a combination of 7t15-21s and anti-TF antibodies.

[0318] Figure 177 This figure shows the changes in surface phenotype of NK cells expanded by 7t15-21s+anti-TF antibody.

[0319] Figure 178 is a schematic diagram and a set of graphs showing the persistence of 7t15-21s and anti-antibody-expanded NK cells in NSG mice after treatment with 7t15-21, TGFRt15-TGFRs, or 2t2.

[0320] Figure 179A and 179B A set of images and graphs showing that NK cells expanded with 7T15-21S+ anti-TF antibodies kill chemically induced senescent human fibroblasts.

[0321] Figures 180A-180C A set of images and a set of graphs showing that NK cells expanded with 7T15-21S+ anti-TF antibodies kill UV-induced senescent human fibroblasts.

[0322] Figure 181 Shown is the proliferation of NK cells after stimulation with 7t15-21s, 7t15-21s+anti-TF antibody (IgG1), or 7t15-21s+anti-TF antibody (IgG4).

[0323] Figure 182 Shows NK cell expansion induced by 7t15-21s+anti-TF antibody (IgG1) and 7t15-21s137L (short version)+anti-TF antibody (IgG1).

[0324] Figure 183 Shown is the clearance of Daudi tumor cells in NSG mice after treatment with NK cells activated by a complex of 7t15-21s and anti-TF antibody (IgG1).

[0325] Figures 184A-184E ApoE-positive mice fed a Western diet and treated with 2t2 or TGFRt15-TGFRs - / - Improvement of skin texture in mice. Figure 184A ApoE was fed a normal diet one week after PBS treatment. - / - Representative photographs of mice. Figure 184B Western diet-fed ApoE was taken one week after PBS treatment. - / - Representative photographs of mice. Figure 184C Shown is a Western diet-fed ApoE taken one week after TGFRt15-TGFRs treatment - / - Representative photographs of mice. Figure 184D Western diet-fed ApoE was taken one week after 2t2 treatment - / - Representative photographs of mice. Figure 184E Western diet-fed ApoE was taken one week after treatment with 21t15-TGFRs. - / - Representative photographs of mice.

[0326] Figure 185 Graph showing activation of Factor X (FX) following treatment with single-chain or multi-chain chimeric polypeptides.

[0327] Figure 186 Shown are the clotting times of buffer solutions containing different concentrations of Innovin in a prothrombin time (PT) test.

[0328] Figure 187 Shown are the clotting times of the multi-chain chimeric polypeptides in the PT assay.

[0329] Figure 188 Shown are the clotting times of the multi-chain chimeric polypeptides in the PT assay when mixed with 32DB cells.

[0330] Figure 189 Shown are the clotting times of the multi-chain chimeric polypeptides in the PT assay when mixed with human PBMCs.

[0331] Figure 190 Binding of 7t15-21s137L (long version) and 7t15-21s137L (short version) to CD137 (4.1BB) is shown.

[0332] Figures 191A-191DDetection of IL7, IL21, IL15, and 4.1BBL in 7t15-21s137L (long version) using ELISA by corresponding antibodies is shown.

[0333] Figure 192 Shown are IL-15 activities of 7t15-21s137L (long version) and 7t15-21s137L (short version) as assessed by IL2Rαβγ CTLL2 cell proliferation assay.

[0334] Figure 193 A-193C showed that in response to 2t2 or IL2 treatment, CD4 + CD25 hi T reg cells, CD4 + CD25 - T con cells or CD8 + T con pStat5a responses in human blood lymphocytes. Figure 193 A shows CD4 + CD25 hi T reg pSTAT5 responses in cells. Figure 193 B shows CD4 + CD25 - T con pSTAT5 responses in cells. Figure 193 C shows CD8 + T con pSTAT5 responses in cells.

[0335] Figure 194 is a graph showing the difference in the percentage of DNA demethylation in NK cells from two different donors relative to unexposed NK cells after expansion with 7t15-21s + anti-tissue factor (TF) antibody (IgG1)) (50 nM). DETAILED DESCRIPTION

[0336] Provided herein are a variety of single-chain chimeric polypeptides and multi-chain chimeric polypeptides, methods of use thereof, and kits comprising the polypeptides. In some embodiments, single-chain and multi-chain chimeric polypeptides include a joint domain located between two target binding domains. In some embodiments, the joint domain is or includes a soluble tissue factor domain. In some embodiments, the joint domain can be recognized by a homologous IgG1 antibody (e.g., a monoclonal antibody). In some embodiments, the single-chain or multi-chain chimeric polypeptides provided herein can be used to stimulate immune cells, induce or increase immune cell proliferation, induce differentiation of immune cells, or treat subjects in need thereof (e.g., subjects suffering from cancer or a disease or condition associated with aging).

[0337] Single-chain chimeric polypeptide

[0338] Some embodiments of any of the methods described herein include using any of the single-chain chimeric polypeptides described herein. Some embodiments of any of the kits described herein include any of the single-chain chimeric polypeptides described herein. The single-chain chimeric polypeptides provided herein include a first target binding domain, a linker domain, and a second target binding domain, wherein the first target binding domain and the second target binding domain are each independently selected from the group consisting of a soluble interleukin or cytokine protein, an antigen binding domain, and a soluble interleukin or cytokine receptor and a ligand for a costimulatory molecule.

[0339] In some examples of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide can have a total length of about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids,about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 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 440 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids,about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, 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 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 about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids,about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, 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 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 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 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 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids acid, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids,about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 4 00 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids,about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids,about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids,about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids , about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids. ,

[0340] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any exemplary target binding domain described herein or known in the art) and the linker domain (e.g., any exemplary linker described herein) are directly adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the first target binding domain (e.g., any exemplary target binding domain described herein or known in the art) and the linker domain (e.g., any exemplary linker domain described herein). In some embodiments of any of the single-chain chimeric polypeptides described herein, the linker domain (e.g., any exemplary linker domain described herein) and the second target binding domain (e.g., any exemplary target binding domain described herein or known in the art) are directly adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker domains described herein) and a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0341] 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 directly adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary target binding domains 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,

[0342] The second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the linker domain (e.g., any of the exemplary linker domains described herein) are directly adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the linker domain (e.g., any of the exemplary linker domains described herein or known in the art).

[0343] Non-limiting aspects of these chimeric polypeptides, nucleic acids, vectors, cells and methods are described below and can be used in any combination without limitation. Other aspects of these chimeric polypeptides, nucleic acids, vectors, cells and methods are known in the art.

[0344] Multi-chain chimeric polypeptides

[0345] Some embodiments of any of the methods described herein comprise use of any of the multi-chain chimeric polypeptides described herein. Some embodiments of any of the kits described herein comprise any of the multi-chain chimeric polypeptides described herein.

[0346] The multi-chain chimeric polypeptides provided herein include: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a linker domain; and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated through the binding of the first domain and the second domain of the pair of affinity domains.

[0347] In some examples of any of the multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide can each independently be from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, from about 50 amino acids to about 700 amino acids, from about 50 amino acids to about 800 amino acids. 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, 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 440 amino acids, about 150 amino acids to about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids,about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, 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 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 about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids,about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 amino acids amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, 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 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 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 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 amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids acid, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about 440 amino acids, about 360 amino acids to about 420 amino acids, about 360 amino acids to about 400 amino acids, about 360 amino acids to about 380 amino acids, about 380 amino acids to about 3000 amino acids, about 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids,about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 4 00 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids,about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids,about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids,about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids , about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids. , Figure 1 and Figure 2 Figures depicting exemplary multi-chain chimeric polypeptides provided herein.

[0348] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any first target binding domain described herein) and the linker domain (e.g., any exemplary linker domain described herein) are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the first target binding domain (e.g., any exemplary first target binding domain described herein) and the linker domain (e.g., any exemplary linker domain described herein).

[0349] In some embodiments of any of the multi-chain chimeric polypeptides described herein, a linker domain (e.g., any exemplary linker domain described herein) and a first domain of a pair of affinity domains (e.g., any exemplary first domain of any pair of exemplary affinity domains described herein) are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker domain described herein) between the linker domain (e.g., any exemplary linker domain described herein) and the first domain of a pair of affinity domains (e.g., any exemplary first domain of any pair of exemplary affinity domains described herein).

[0350] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of a pair of affinity domains (e.g., any exemplary second domain of any pair of exemplary affinity domains described herein) and the second target binding domain (e.g., any exemplary second target binding domain described herein) are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein,

[0351] The second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of a pair of affinity domains (e.g., any of the exemplary second domains of any pair of exemplary affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein).

[0352] Tissue Factor

[0353] 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: P13726). The cytoplasmic tail is located at Ser 253 and Ser 258 Contains two phosphorylation sites at Cys 245 There is an S-palmitoylation site at the cytoplasmic domain. No deletion or mutation of the cytoplasmic domain has been found to affect the coagulation activity of tissue factor. 245 There is an S-palmitoylation site at Cys 245 Located at the amino acid end of the intracellular domain and close to the membrane surface. The tissue factor transmembrane domain is composed of a single spanning α-helix.

[0354] 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, allowing the extracellular domain of tissue factor to decouple from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping β-sheets, with the top fold containing three antiparallel β-strands and the bottom fold containing four β-strands. The β-strands are connected by β-loops between chains βA and βB, βC and βD, and βE and βF, which are conformationally conserved in both modules. There are three short α-helical segments connecting the β-strands. A unique feature of tissue factor is the 17-amino acid β-hairpin between chains β10 and β11, which is not a common component of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is not present in the C-terminal domain and is unique to tissue factor. This fibronectin type III domain structure is characteristic of the immunoglobulin-like family of proteins and is conserved in a wide variety of extracellular proteins.

[0355] Once zymogen FVII is bound to tissue to form the active tissue factor-FVIIa complex, it is rapidly converted to FVIIa by limited proteolysis. FVIIa circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII) and can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa at the tissue factor-FVIIa complex greatly enhances the enzymatic activity of FVIIa: the rate of hydrolysis of small chromogenic peptide substrates is increased by approximately 20 to 100 times, and the rate of activation of the natural macromolecular substrates FIX and FX is increased by nearly a million times. Consistent with allosteric activation of the FVIIa active site upon binding to tissue factor, formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., when phosphatidyl-L-serine is exposed at the membrane surface) is regulated by Ca 2+ The approximately million-fold overall increase in FX activation by the tissue factor-FVIIa-phospholipid complex relative to free FVIIa is a key regulatory point in the coagulation cascade.

[0356] FVII is a single-chain polypeptide of approximately 50 kDa, consisting of 406 amino acid residues, with an N-terminal γ-carboxyglutamic acid (GLA)-rich domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII is a protein expressed in a short linker region between the EGF2 and protease domains. 154 -Arg 152 FVIIa is activated by specific proteolytic cleavage of the light chain and heavy chain through Cys 135 and Cys 262 FVIIa is held together by a single disulfide bond. 2+ The C-terminus of the GLA domain is immediately adjacent to an aromatic stack and two EGF domains. The aromatic stack connects GLA to the binding of a single Ca 2+ ion of EGF1 domain. Occupying this Ca 2+ 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 2+ The binding of ions is crucial for its catalytic activity. Proteolytic activation of FVIIa by FVII releases Ile 153 The newly formed amino terminus folds back and inserts into the activation pocket, aligning with Asp 343The carboxylate of FVIIa forms a salt bridge to generate an oxyanion pore. The formation of this salt bridge is crucial for FVIIa activity. However, after proteolytic activation, oxyanion pore formation does not occur in free FVIIa. As a result, FVIIa circulates in a zymogen-like state, which is poorly recognized by plasma protease inhibitors, causing it to circulate with a half-life of approximately 90 minutes.

[0357] Tissue factor-mediated positioning of the FVIIa active site above the membrane surface is important for FVIIa to be oriented toward its cognate substrate. Free FVIIa adopts a stable extended structure when bound to the membrane, with its active site positioned approximately After FVIIa binds to tissue factor, the FVa active site is relocated closer to the membrane. This regulation helps the FVIIa catalytic triad to properly align with the target substrate cleavage site. Using FVIIa without the GLA domain, it has been shown that the active site remains located at a similar distance above the membrane, demonstrating that tissue factor can fully support the positioning of the FVIIa active site even in the absence of FVIIa-membrane interaction. Other data show that tissue factor supports full FVIIa proteolytic activity as long as the tissue factor extracellular domain is tethered to the membrane surface in some way. However, raising the FVIIa active site above the membrane surface by more than 100 μm will result in a significant decrease in the FVIIa active site. It significantly reduces the ability of the tissue factor-FVIIa complex to activate FX, but does not reduce the amidolytic activity of tissue factor-FVIIa.

[0358] Alanine scanning mutagenesis has been used to assess the effects of specific amino acid side chains in the extracellular domain of tissue factor on the interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitution identified a limited number of residue positions where alanine replacement caused a 5- to 10-fold decrease in affinity for FVIIa binding. Most of these residue side chains were found to be fully exposed to the solvent in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand binding site is located in a broad region at the boundary between the two modules. In the C module, the residue Arg located on the prominent BC loop is located in the C module. 135 and Phe 140 Provides independent contact with FVIIa. 133 Located at the base of the finger-like structure and squeezed into the crack between the two modules. This provides 20 、Thr 60 、Asp 58and Ile 22 The continuity of the main cluster of important binding residues. 60 It is only partially exposed to solvent and likely plays a local structural role rather than making significant contacts with the ligand. The binding site extends to the concave surface of the intermodule angle, which involves Glu 24 and Gln 110 , and possibly involving more distant residues Val 207 The binding region extends from Asp58 to the 48 、Lys 46 、Gln 37 、Asp 44 and Trp 45 The convex surface area formed. 45 and Asp 44 does not interact independently with FVIIa, indicating that Trp 45 The effect of mutation at position 1 may reflect the effect of this side chain on the adjacent Asp 44 and Gln 37 The interaction region also includes two surface-exposed aromatic residues Phe 76 and Tyr 78 , which forms part of a hydrophobic cluster in the N module.

[0359] The known physiological substrates of tissue factor-FVIIa are FVII, FIX and FX and certain protease-activated receptors. Mutational analysis has identified many residues that, when mutated, support full FVIIa amidolytic activity on small peptide substrates, but lack the ability to support activation of macromolecular substrates (i.e., FVII, FIX and FX) (Ruf et al., Journal of Biol. Chem. 267(31):22206-22210, 1992; Ruf et al., Journal of Biol. Chem. 267(9):6375-6381, 1992; Huang et al., Journal of Biol. Chem. 271(36):21752-21757, 1996; Kirchhofer et al., Biol. Chem. 39(25):7380-7387, 2000). It has been shown that the tissue factor loop region at residues 159-165 and residues in or near this flexible loop are crucial for the proteolytic activity of the tissue factor-FVIIa complex. This defines a region outside the proposed substrate binding site of tissue factor that is quite distant from the FVIIa active site. Substitution of the glycine residue with the slightly larger alanine residue significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility provided by glycine is crucial for the loop of residues 159-165 that is used for tissue factor macromolecular substrate recognition.

[0360] It was also shown that the residue Lys 165and Lys 166 Important for substrate recognition and binding. Mutation of any of these residues to alanine results in a significant reduction in tissue factor cofactor function. In most tissue factor-FVIIa structures, Lys 165 and Lys 166 With their backs to each other, Lys 165 points to FVIIa, while Lys 166 Pointing to the region outside the substrate binding site in the crystal structure. Lys 165 and Gla 35 The putative salt bridge formation between FIX and FX supports the notion that tissue factor interacts with the GLA domain of FVIIa to regulate substrate recognition. These results suggest that the C-terminal portion of the extracellular domain of tissue factor directly interacts with the GLA domains of FIX and FX, and possibly the adjacent EGF1 domain, and that the presence of the FVIIa GLA domain can modulate these interactions directly or indirectly.

[0361] Exemplary linker domains and IgG1 antibody constructs

[0362] In some examples of any method described herein, compositions or test kits, the joint domain can be or include any antigen identified by a homologous IgG1 antibody (e.g., monoclonal antibody). The difference between IgG1 antibodies and other IgG classes of antibodies is their constant region, especially hinge region and upper CH2 domain (see, e.g., Vadarsson et al., IgG subclasses and allotypes: From structure to effector function (IgG Subclasses and Allotypes:From Structure to Effector Functions), " Frontiers in Immunology (Front Immunol.) ", 5, Chapter 520, 2014). Human IgG1 is the only IgG subclass known to bind to human CD16a and activate the signaling of human CD16a.

[0363] Any of a plurality of linker domains can be used in single-chain chimeric polypeptides and multi-chain chimeric polypeptides provided herein. In certain embodiments, the linker domain is recognized by an antibody (e.g., an IgG1 antibody) or an antibody fragment. In certain embodiments, the linker domain is an antibody kappa light chain recognized by a homologous anti-κ light chain IgG1 antibody. In certain embodiments, the linker domain is an antibody lambda light chain recognized by a homologous anti-λ light chain human IgG1 antibody. Multiple kappa light chains and lambda light chains are known in the art (see, e.g., Smith et al., Antigen Nature and Complexity Influence Human Antibody Light Chain Usage and Specificity, Vaccine, 34(25):2813-2820, 2016). In some embodiments, the linker domain is or comprises a human polypeptide (e.g., a human kappa light chain of an antibody or a human lambda light chain of an antibody) that is recognized by a homologous human IgG1 antibody (e.g., a monoclonal antibody). In some embodiments, the homologous human IgG1 antibody comprises at least two antigen binding domains, and each antigen binding domain specifically binds to the linker domain. In some embodiments, the homologous human IgG1 antibody comprises at least two antigen binding domains, and only a subset (e.g., one) of the antigen binding domains specifically binds to the linker domain. In some embodiments, the linker domain can be any one of the soluble tissue factor domains described herein.

[0364] In some embodiments of any of the methods, compositions and kits described herein, the IgG1 antibody construct can be an IgG1 antibody (e.g., a monoclonal or polyclonal IgG1 antibody that specifically binds to a joint domain). In some embodiments of any of the methods, compositions and kits described herein, the IgG1 antibody construct can be an antibody or antibody fragment that includes an IgG1 Fc region (e.g., a human IgG1 Fc region) and specifically binds to a joint domain. As known in the art, the IgG1 Fc region binds to CD16a (FcRγIII) (e.g., human CD16a) and induces its intracellular signaling. In some embodiments, the IgG1 antibody construct can be a single-chain or multi-chain polypeptide that includes an Fc region that can specifically bind to CD16a (FcRγIII) (e.g., human CD16a) and can induce its intracellular signaling in natural killer cells (e.g., human natural killer cells), and specifically binds to a joint domain. In some embodiments, the IgG1 antibody construct can be a single-chain or multi-chain polypeptide comprising an Fc region that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical) to a wild-type IgG1 Fc domain (e.g., a wild-type human IgG1 Fc domain, e.g., SEQ ID NO: 96) and is capable of specifically binding to CD16a (FcRγIII) (e.g., human CD16a) and capable of inducing its intracellular signaling in natural killer cells (e.g., human natural killer cells). In some embodiments of any of the methods, compositions, or kits described herein, the IgG1 antibody construct can be an antibody or antibody fragment that specifically binds to a linker domain and includes a non-IgG1 Fc region (e.g., an IgG2, IgG3, or IgG4 Fc region) that has been altered (e.g., replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in a wild-type non-IgG1 Fc region) such that the non-IgG1 Fc region is able to bind to CD16a (FcRγIII) (e.g., human CD16a) and induce its intracellular signaling in natural killer cells (e.g., human natural killer cells).In some embodiments of any of the methods, compositions, or kits described herein, the IgG1 antibody construct is specifically bound to a linker domain and includes a non-human Fc region (e.g., an Fc region from a non-human antibody) that has been altered (e.g., by replacing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in a wild-type non-human Fc region) such that the non-human Fc region is able to bind to human CD16a (human FcRγIII) and induce its intracellular signaling in natural killer cells (e.g., human natural killer cells).

[0365] Wild-type human IgG1 Fc region (SEQ ID NO: 96)

[0366] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0367] Wild-type human IgG2 Fc region (SEQ ID NO: 97)

[0368] APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGS...

Claims

1. A method for promoting the activation and proliferation of natural killer cells or T cells, the method comprising: contacting natural killer cells or T cells in a liquid medium under conditions that permit activation and proliferation of the natural killer cells or the T cells, the liquid medium comprising: (1) an effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain comprising a sequence having at least 85% identity to SEQ ID NO: 1; and (iii) a first domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 39; and (b) a second chimeric polypeptide comprising: (i) a second domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 10; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate by binding of the first domain and the second domain of the pair of affinity domains; and (I) the first target binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41, and the second target binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35; or (II) the first target binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35, and the second target binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41; and (2) an effective amount of an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to the soluble tissue factor domain.

2. The method according to claim 1, wherein the NK cells or T cells are previously obtained from a subject.

3. The method according to claim 1 or 2, wherein the NK cells or T cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor.

4. The method according to claim 1 or 2, wherein the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells or the T cells after the contacting step.

5. A method for increasing the glucose consumption of natural killer cells, the method comprising: contacting natural killer cells in a liquid medium under conditions that permit glucose consumption of the natural killer cells, the liquid medium comprising: (1) an effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain comprising a sequence having at least 85% identity to SEQ ID NO: 1; and (iii) The first domain of a pair of affinity domains, which comprises a sequence having at least 85% identity with SEQ ID NO: 39; (b) A second chimeric polypeptide, which comprises: (i) The second domain of a pair of affinity domains, which comprises a sequence having at least 85% identity with SEQ ID NO: 10; and (ii) A second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (I) The first target-binding domain comprises a sequence having at least 85% identity with SEQ ID NO: 41, and the second target-binding domain comprises a first sequence having at least 85% identity with SEQ ID NO: 33 and a second sequence having at least 85% identity with SEQ ID NO: 35; or (II) The first target-binding domain comprises a first sequence having at least 85% identity with SEQ ID NO: 33 and a second sequence having at least 85% identity with SEQ ID NO: 35, and the second target-binding domain comprises a sequence having at least 85% identity with SEQ ID NO: 41; and (2) An effective amount of an IgG1 antibody construct, which comprises at least one antigen-binding domain that specifically binds to the soluble tissue factor domain.

6. A method for increasing the oxidative phosphorylation of natural killer cells, the method comprising: contacting natural killer cells in a liquid medium under conditions that permit the oxidative phosphorylation of the natural killer cells, the liquid medium comprising: (1) An effective amount of a multi-chain chimeric polypeptide, which comprises: (a) A first chimeric polypeptide, which comprises: (i) A first target-binding domain; (ii) A soluble tissue factor domain, which comprises a sequence having at least 85% identity with SEQ ID NO: 1; and (iii) The first domain of a pair of affinity domains, which comprises a sequence having at least 85% identity with SEQ ID NO: 39; (b) A second chimeric polypeptide, which comprises: (i) The second domain of a pair of affinity domains, which comprises a sequence having at least 85% identity with SEQ ID NO: 10; and (ii) A second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (I) The first target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41, and the second target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35; or (II) The first target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35, and the second target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41; and (2) An effective amount of an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to the soluble tissue factor domain.

7. A method of increasing the aerobic glycolysis of natural killer cells, the method comprising: contacting natural killer cells in a liquid medium under conditions that permit aerobic glycolysis of the natural killer cells, the liquid medium comprising: (1) An effective amount of a multi-chain chimeric polypeptide comprising: (a) A first chimeric polypeptide comprising: (i) A first target-binding domain; (ii) A soluble tissue factor domain comprising a sequence having at least 85% identity to SEQ ID NO: 1; and (iii) A first domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 39; (b) A second chimeric polypeptide comprising: (i) A second domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 10; and (ii) A second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate by binding of the first domain and the second domain of the pair of affinity domains; and (I) The first target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41, and the second target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35; or (II) The first target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35, and the second target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41; and (2) An effective amount of an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to the soluble tissue factor domain.

8. A method of increasing the extracellular acidification rate (ECAR) of natural killer cells, the method comprising: Contact natural killer cells in a liquid medium under conditions that permit extracellular acidification of the natural killer cells, the liquid medium comprising: (1) An effective amount of a multichain chimeric polypeptide comprising: (a) A first chimeric polypeptide comprising: (i) A first target-binding domain; (ii) A soluble tissue factor domain comprising a sequence having at least 85% identity to SEQ ID NO: 1; and (iii) A first domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 39; (b) A second chimeric polypeptide comprising: (i) A second domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 10; and (ii) A second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (I) The first target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41, and the second target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35; or (II) The first target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35, and the second target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41; and (2) An effective amount of an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to the soluble tissue factor domain.

9. A method of increasing the mitochondrial oxygen consumption rate of natural killer cells, the method comprising: Contact natural killer cells in a liquid medium under conditions that permit mitochondrial oxygen consumption of the natural killer cells, the liquid medium comprising: (1) An effective amount of a multichain chimeric polypeptide comprising: (a) A first chimeric polypeptide comprising: (i) A first target-binding domain; (ii) A soluble tissue factor domain comprising a sequence having at least 85% identity to SEQ ID NO: 1; and (iii) A first domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 39; (b) A second chimeric polypeptide comprising: (i) A second domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 10; and (ii) A second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains; and (I) The first target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41, and the second target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35; or (II) The first target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35, and the second target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41; and (2) An effective amount of an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to the soluble tissue factor domain.

10. A method of inducing natural killer cells to differentiate into memory or memory-like natural killer cells, the method comprising: contacting natural killer cells in a liquid culture medium comprising: (1) An effective amount of a multi-chain chimeric polypeptide comprising: (a) A first chimeric polypeptide comprising: (i) A first target-binding domain; (ii) A soluble tissue factor domain comprising a sequence having at least 85% identity to SEQ ID NO: 1; and (iii) The first domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 39; (b) A second chimeric polypeptide comprising: (i) The second domain of a pair of affinity domains comprising a sequence having at least 85% identity to SEQ ID NO: 10; and (ii) A second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate by binding of the first domain and the second domain of the pair of affinity domains; and (I) The first target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41, and the second target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35; or (II) The first target-binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35, and the second target-binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41; and (2) An effective amount of an IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to the soluble tissue factor domain.

11. The method according to any one of claims 5-10, wherein the natural killer cells are previously obtained from a subject.

12. The method according to any one of claims 5-10, wherein the natural killer cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor.

13. The method according to any one of claims 5-10, wherein the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the natural killer cells after the contacting step.

14. The method according to any one of claims 1, 2 and 5-10, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

15. The method according to any one of claims 1, 2 and 5-10, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

16. The method according to any one of claims 1, 2 and 5-10, wherein the soluble tissue factor domain and the first domain of a pair of affinity domains are directly adjacent to each other in the first chimeric polypeptide.

17. The method according to any one of claims 1, 2 and 5-10, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of a pair of affinity domains in the first chimeric polypeptide.

18. The method according to any one of claims 1, 2 and 5-10, wherein the second domain of the pair of affinity domains in the second chimeric polypeptide and the second target binding domain are directly adjacent to each other.

19. The method according to any one of claims 1, 2 and 5-10, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target binding domain in the second chimeric polypeptide.

20. The method according to claim 1, wherein the soluble human tissue factor domain does not stimulate blood coagulation.

21. The method according to any one of claims 1, 2 and 5-10, wherein the contacting step is carried out for a period of 2 hours to 20 days.

22. The method according to any one of claims 1, 2 and 5-10, wherein the liquid medium contains the multichain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of 0.5:1 to 2:

1.

23. The method according to any one of claims 1, 2 and 5-10, wherein: the first target binding domain comprises SEQ ID NO:41; the soluble tissue factor domain comprises SEQ ID NO:1; the first domain of the pair of affinity domains comprises SEQ ID NO:39; the second target binding domain comprises a first sequence containing SEQ ID NO:33 and a second sequence containing SEQ ID NO:35; and the second domain of the pair of affinity domains comprises SEQ ID NO:

10.

24. The method according to any one of claims 1, 2 and 5-10, wherein: the first chimeric polypeptide comprises SEQ ID NO:116; and The second chimeric polypeptide comprises SEQ ID NO:

120.

25. The method according to any one of claims 1, 2, and 5-10, wherein: The first target binding domain comprises a first sequence containing SEQ ID NO: 33 and a second sequence containing SEQ ID NO: 35; The soluble tissue factor domain comprises SEQ ID NO: 1; The first domain of the pair of affinity domains comprises SEQ ID NO: 39; The second target binding domain comprises SEQ ID NO: 41; and The second domain of the pair of affinity domains comprises SEQ ID NO:

10.

26. The method according to any one of claims 1, 2, and 5-10, wherein The first chimeric polypeptide comprises amino acids 19 to 869 of SEQ ID NO: 192; and The second chimeric polypeptide comprises amino acids 19 to 240 of SEQ ID NO:

191.

27. A kit, comprising: (1) A multichain chimeric polypeptide, comprising: (a) A first chimeric polypeptide, comprising: (i) A first target binding domain; (ii) A soluble tissue factor domain, comprising a sequence having at least 85% identity to SEQ ID NO: 1; and (iii) The first domain of a pair of affinity domains, comprising a sequence having at least 85% identity to SEQ ID NO: 39; (b) A second chimeric polypeptide, comprising: (i) The second domain of a pair of affinity domains, comprising a sequence having at least 85% identity to SEQ ID NO: 10; and (ii) A second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate by the binding of the first domain and the second domain of the pair of affinity domains; and (I) The first target binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41, and the second target binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35; or (II) The first target binding domain comprises a first sequence having at least 85% identity to SEQ ID NO: 33 and a second sequence having at least 85% identity to SEQ ID NO: 35, and the second target binding domain comprises a sequence having at least 85% identity to SEQ ID NO: 41; and (2) An IgG1 antibody construct, comprising at least one antigen binding domain that specifically binds to the soluble tissue factor domain.

28. The kit according to claim 27, wherein: The first target binding domain comprises SEQ ID NO: 41; The soluble tissue factor domain comprises SEQ ID NO: 1; The first domain of the pair of affinity domains comprises SEQ ID NO: 39; The second target binding domain comprises a first sequence containing SEQ ID NO: 33 and a second sequence containing SEQ ID NO: 35; and The second domain of the pair of affinity domains comprises SEQ ID NO:

10.

29. The kit according to claim 27, wherein: The first chimeric polypeptide comprises SEQ ID NO: 116; and The second chimeric polypeptide comprises SEQ ID NO:

120.

30. The kit according to claim 27, wherein the first target binding domain comprises: A first sequence containing SEQ ID NO: 33 and a second sequence containing SEQ ID NO: 35; The soluble tissue factor domain comprises SEQ ID NO: 1; The first domain of the pair of affinity domains comprises SEQ ID NO: 39; The second target binding domain comprises SEQ ID NO: 41; and The second domain of the pair of affinity domains comprises SEQ ID NO:

10.

31. The kit according to claim 27, wherein: The first chimeric polypeptide comprises amino acids 19 to 869 of SEQ ID NO: 192; and The second chimeric polypeptide comprises amino acids 19 to 240 of SEQ ID NO: 191.

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