Multi-chain chimeric polypeptides and uses thereof

Abstract

Provided herein are multi-chain chimeric polypeptides and their use for the treatment of cancer, diseases or conditions associated with aging, and / or infectious diseases. Further provided are methods for stimulating an immune cell, inducing or increasing proliferation of an immune cell, and / or inducing differentiation of an immune cell into a memory-like immune cell by contacting the immune cell with the multi-chain chimeric polypeptides disclosed herein.

Description

Cross-reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 594,637, filed October 31, 2023, pursuant to 35 USC §119(e). The entire disclosure is incorporated herein by reference. References to sequence lists

[0002] This application contains a sequence list submitted electronically as an XML file, which is incorporated herein by reference in its entirety. The XML file, created on October 28, 2024, is named “8774HCW-17.xml” and has a size of 59,305 bytes. Technical Field

[0003] This disclosure relates to the field of biotechnology, and more specifically, to multi-chain chimeric peptides and methods for treating cancer, age-related diseases or conditions, and / or infectious diseases. This document further envisions methods for stimulating immune cells, inducing or increasing the proliferation of immune cells, and / or inducing the differentiation of immune cells into memory-like immune cells by contacting immune cells with the multi-chain chimeric peptides disclosed herein. Background Technology

[0004] Tissue factor (TF), an integrated membrane glycoprotein of approximately 46 kDa and 263 amino acids, and a trigger protein of the extrinsic coagulation pathway, is a major initiator of coagulation in vivo. TF is normally not in contact with circulating blood, but it initiates the coagulation cascade upon exposure to circulating coagulation serine protease factors. Vascular injury exposes subendothelial cells expressing TF, leading to the formation of a calcium-dependent, high-affinity complex with pre-existing plasma factor VIIa (FVIIa). The binding of the serine protease FVIIa to TF promotes rapid cleavage of FX to FXa and rapid cleavage of FIX to FIXa. The resulting FXa has insufficient proteolytic activity and an inefficient membrane surface to convert small amounts of prothrombin into thrombin. The thrombin generated from FXa initiates platelet activation and activates trace amounts of pro-cofactors (factor V (FV) and factor VIII (FVIII)) into active cofactors (factor Va (FVa) and factor VIIIa (FVIIIa)). FIXa complexes with FVIIIa on the platelet surface to form an intrinsic tenase complex, leading to the rapid generation of FXa. FXa then complexes with FVa to form a prothrombinase complex on the activated platelet surface, which results in the rapid cleavage of prothrombin into thrombin.

[0005] In addition to the tissue factor-FVIIa complex, a recent study showed that the tissue factor-FVIIa-FXa complex can also activate FVIII, thereby providing additional levels of FVIIIa in the initial phase. The extrinsic pathway is crucial in initiating coagulation by activating a limited amount of thrombin, while the intrinsic pathway maintains coagulation through a sharp amplification of the initial signal.

[0006] Many tissue factors expressed on the cell surface are "encrypted" and must be "decrypted" to fully participate in coagulation. Currently, the mechanism of this "decryption" is unclear, but the exposure of anionic phospholipids plays a crucial role. Healthy cells actively isolate anionic phospholipids (such as phosphatidylserine (PS)) into the inner lobe of the plasma membrane. Cell damage, activation, or cytoplasmic calcium... 2+ Upon increasing levels, this bilayer asymmetry disappears, leading to increased PS exposure on the outer leaflets, thereby increasing the specific activity of the cell surface tissue factor-FVIIa complex. PS exposure is known to reduce the apparent Km of FIX and FX activated by the tissue factor-FVIIa complex, but other mechanisms may include conformational rearrangement of tissue factor or tissue factor-FVIIa and subsequent exposure to substrate binding sites. Summary of the Invention

[0007] This document provides multi-chain chimeric polypeptides comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain comprising: (i) a first sequence at least 90% identical to SEQ ID NO: 2, wherein the amino acid at position 32 of SEQ ID NO: 2 is asparagine and the amino acid at position 119 of SEQ ID NO: 2 is alanine; and a second sequence at least 90% identical to SEQ ID NO: 1, wherein the amino acid at position 32 of SEQ ID NO: 2 is asparagine and the amino acid at position 119 of SEQ ID NO: 2 is alanine; (ii) a soluble tissue factor domain comprising a sequence at least 90% identical to SEQ ID NO: 1; and (iii) a first domain of an affinity domain pair comprising a sequence at least 90% identical to SEQ ID NO: 13; and (b) a second chimeric polypeptide comprising: (i) The first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first and second domains of the affinity pair. The second domain contains at least 90% of the sequence identical to SEQ ID NO:11; and (ii) a second target-binding domain, the second target-binding domain containing: a first sequence identical to at least 90% of SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine; and a second sequence identical to at least 90% of SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first and second domains of the affinity pair.

[0008] In some embodiments of any multi-chain chimeric peptide described herein, the first target-binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric peptide. In some embodiments of any multi-chain chimeric peptide described herein, the first chimeric peptide further includes a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric peptide.

[0009] In some embodiments of any multi-chain chimeric peptide described herein, the first domains of the soluble tissue factor domain and the affinity domain pair are directly adjacent to each other in the first chimeric peptide. In some embodiments of any multi-chain chimeric peptide described herein, the first chimeric peptide further includes a linker sequence between the first domains of the soluble tissue factor domain and the affinity domain pair in the first chimeric peptide.

[0010] In some embodiments of any multi-chain chimeric peptide described herein, the second domain of the affinity pair and the second target-binding domain are directly adjacent to each other in the second chimeric peptide. In some embodiments of any multi-chain chimeric peptide described herein, the second chimeric peptide further includes a linker sequence between the second domain of the affinity pair and the second target-binding domain in the second chimeric peptide.

[0011] In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide further comprises one or more additional target-binding domains.

[0012] In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide further comprises one or more additional target-binding domains.

[0013] In some embodiments of any multi-chain chimeric polypeptide described herein, the first target-binding domain comprises at least 80% of the sequence identical to SEQ ID NO:4. In some embodiments of any multi-chain chimeric polypeptide described herein, the first target-binding domain comprises at least 90% of the sequence identical to SEQ ID NO:4. In some embodiments of any multi-chain chimeric polypeptide described herein, the first target-binding domain comprises the sequence of SEQ ID NO:4.

[0014] In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide comprises at least 80% of the sequence identical to SEQ ID NO:6. In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide comprises at least 90% of the sequence identical to SEQ ID NO:6. In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO:6. In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO:7.

[0015] In some embodiments of any multi-chain chimeric polypeptide described herein, the second target-binding domain comprises at least 80% of the sequence identical to SEQ ID NO:4. In some embodiments of any multi-chain chimeric polypeptide described herein, the second target-binding domain comprises at least 90% of the sequence identical to SEQ ID NO:4. In some embodiments of any multi-chain chimeric polypeptide described herein, the second target-binding domain comprises the sequence of SEQ ID NO:4.

[0016] In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide comprises at least 80% of the sequence identical to SEQ ID NO:5. In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide comprises at least 80% of the sequence identical to SEQ ID NO:6.

[0017] In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide comprises at least 90% identical sequence to SEQ ID NO:5. In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide comprises the sequence of SEQ ID NO:5. In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO:6.

[0018] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide comprises the sequence of SEQ ID NO:8.

[0019] This document also provides compositions comprising any of the multi-chain chimeric polypeptides described herein. In some embodiments of any of the compositions described herein, the compositions are pharmaceutical compositions.

[0020] This document also provides kits containing at least one dose of any of the compositions described herein.

[0021] This document also provides methods and / or uses of the compositions disclosed herein for stimulating immune cells, including contacting immune cells with an effective amount of any of the multi-chain chimeric polypeptides or any of the compositions described herein.

[0022] This document also provides methods and / or uses of the compositions disclosed herein for inducing or increasing the proliferation of immune cells, including contacting immune cells with an effective amount of any multi-chain chimeric polypeptide or any composition described herein.

[0023] This document also provides methods and / or uses of the compositions disclosed herein for inducing immune cells to differentiate into memory or memory-like immune cells, including contacting immune cells with an effective amount of any multi-chain chimeric polypeptide or any composition described herein.

[0024] In some embodiments of any method and / or use of the compositions described herein, immune cells are contacted in vitro. In some embodiments of any method and / or use of the compositions described herein, immune cells are contacted in vivo.

[0025] In some embodiments of any method and / or use of the compositions described herein, the immune cells are selected from the group consisting of: immature thymocytes, peripheral blood lymphocytes, primary T cells, pluripotent Th cell precursors, lymphoid progenitor cells, Treg cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8+ T cells, CD4+ T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells.

[0026] In some embodiments of any method and / or use of the compositions described herein, the immune cells have previously been genetically modified to express chimeric antigen receptors or recombinant T-cell receptors.

[0027] This document also provides methods and / or uses of the compositions disclosed herein for killing cancer cells, infected cells, or senescent cells in a subject in need, including administering a therapeutically effective amount of any of the multi-chain chimeric peptides or any of the compositions described herein to the subject.

[0028] This document also provides methods and / or uses of the compositions disclosed herein for treating a subject in need, including administering a therapeutically effective amount of any of the multi-chain chimeric peptides or any of the compositions described herein to the subject.

[0029] In some embodiments of any method and / or use of the compositions described herein, the subject has been identified or diagnosed with cancer, an age-related disease or condition, or an infectious disease. In some embodiments of any of the methods described herein, the cancer is selected from the group consisting of: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal 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.

[0030] In some embodiments of any method and / or use of the compositions described herein, the age-related diseases or conditions are selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of elasticity of lung tissue, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

[0031] In some embodiments of any method and / or use of the compositions 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, human papillomavirus and influenza virus.

[0032] This document also provides methods and / or uses of the compositions disclosed herein for killing naturally occurring and / or treatment-induced senescent cells or reducing the number of such senescent cells in a subject, including administering a therapeutically effective amount of any of the multi-chain chimeric peptides or any of the compositions described herein to the subject.

[0033] This document also provides methods and / or uses of the compositions disclosed herein for reducing the level and / or activity of one or more SASP factors derived from naturally occurring and / or treatment-induced senescent cells in a subject, including administering to the subject a therapeutically effective amount of any of the multi-chain chimeric peptides or any of the compositions described herein.

[0034] In some embodiments of any method and / or use of the compositions described herein, the subject has been previously diagnosed or identified as having an age-related disease or inflammatory condition. In some embodiments of any method described herein, the age-related disease is inflammatory aging-related.

[0035] In some embodiments of any method and / or use of the compositions described herein, the age-related diseases are selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, inflammatory bowel disease, intervertebral disc degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung elasticity, age-related macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegenerative diseases, stroke, cancer, dementia, vascular disease, susceptibility to infection, chronic inflammation, and renal dysfunction.

[0036] In some embodiments of any method and / or use of the compositions described herein, the age-related disease is a cancer selected from the group consisting of: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal 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.

[0037] In some embodiments of any method and / or use of the compositions described herein, the inflammatory disease is selected from the group consisting of: rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS damage, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Crohn's disease, multiple sclerosis, Guillain-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, non-alcoholic steatohepatitis, mood disorders, and cognitive impairment related to cancer treatment.

[0038] In some embodiments of any method and / or use of the compositions described herein, the treatment-induced senescent cells are chemotherapy-induced senescent cells.

[0039] In some embodiments of any method and / or use of the compositions described herein, administration results in a reduction in the number or activity of naturally occurring senescent cells and / or treatment-induced senescent cells in the target tissue of the subject. In some embodiments of any method described herein, the target tissue is selected from the group consisting of: adipose tissue, pancreatic tissue, liver tissue, kidney tissue, lung tissue, heart tissue, vascular system, bone tissue, central nervous system (CNS) tissue, eye tissue, skin tissue, muscle tissue, and secondary lymphatic organ tissue.

[0040] This document also provides nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. Furthermore, this document provides vectors containing any nucleic acid encoding any of the multi-chain chimeric polypeptides described herein.

[0041] This article also provides cells containing any of the nucleic acids or vectors described herein.

[0042] This document also provides methods for generating multi-chain chimeric peptides, including: culturing any of the cells described herein in a culture medium under conditions sufficient to induce the generation of any of the multi-chain chimeric peptides described herein; and recovering the multi-chain chimeric peptides from the cells and / or the culture medium. This document also provides multi-chain chimeric peptides generated using any of the methods described herein.

[0043] This article also provides compositions for treating subjects in need, comprising any of the multi-chain chimeric peptides described herein.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0045] Other features and advantages of the invention will become clear from the following detailed description, drawings and claims. Attached Figure Description

[0046] Figure 1Example diagrams of multi-chain chimeric peptides are shown: (i) a first chimeric peptide comprising a first target-binding domain (A), a soluble tissue factor domain, a first domain of an affinity pair (soluble interleukin IL-15), and an additional target-binding domain (B); and (ii) a second chimeric peptide comprising a second domain of an affinity pair (IL-15 receptor α-sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). A top cartoon depicts the association of the first and second chimeric peptides via the affinity pair. A bottom schematic shows the order of the domains in the first and second chimeric peptides.

[0047] Figure 2 Example diagrams of multi-chain chimeric peptides are shown: (i) a first chimeric peptide comprising a first target-binding domain (A), a soluble tissue factor domain (containing five amino acid substitutions to remove the binding of the soluble tissue factor domain to FVIIa), a first domain of an affinity domain pair (soluble interleukin IL-15, which contains D8N or D8A amino acid substitutions), and an additional target-binding domain (B); and (ii) a second chimeric peptide comprising a second domain of an affinity domain pair (IL-15 receptor α-sushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). A top cartoon depicts the association of the first and second chimeric peptides through the affinity domain pair. A bottom schematic shows the order of the domains in the first and second chimeric peptides. In other embodiments of any multi-chain chimeric polypeptide described herein, the soluble tissue factor domain may comprise or consist of the following: a soluble wild-type human tissue factor domain (comprising or consisting of the following: a continuous sequence within wild-type human tissue factor).

[0048] Figure 3 A schematic diagram of the TGFRt15-TGFR construct is shown.

[0049] Figure 4 Another schematic diagram of the TGFRt15-TGFR construct is shown.

[0050] Figure 5 The following are examples: TGFRt15-TGFR (HCW9218), HCW9228, and TGFR. t15-TGFR The result of TGFβ1 binding of (HCW9238).

[0051] Figure 6 The following are examples: TGFRt15-TGFR (HCW9218), HCW9228, and TGFR. t15-TGFR The result of LAP binding of (HCW9238).

[0052] Figures 7A-7B The following are examples: TGFRt15-TGFR (HCW9218), HCW9228, and TGFR. t15-TGFR Bioactivity of (HCW9238).

[0053] Figures 8A-8B The use of TGFR is shown t15-TGFR Results of 32Dβ cell proliferation assays of (HCW9238) or TGFRt15-TGFR (HCW9218).

[0054] Figure 9 The following are examples: TGFRt15-TGFR (HCW9218), HCW9228, and TGFR. t15-TGFR (HCW9238—arrow in the figure) Comparison of clearance from mouse blood.

[0055] Figure 10 The following data are presented: spleen weight; percentage of immune cells; expression of KLRG1, Ki67, CD25, and granzyme B; and expression of TGFRt15-TGFR (HCW9218), HCW9228, and TGFR. t15-TGFR Mice treated with (HCW9238) were immunostimulated for 96 hours. In each graph showing percentages, the first bar in each group shows the results for CD4+ T cells, the second bar in each group shows the results for CD8+ T cells, and the third bar in each group shows the results for NK cells.

[0056] Figure 11 shows the expansion of antigen-experienced CD8+ T cells in peripheral blood, dLN, and tumors of mice after treatment with HCW9328. Flow cytometry staining of CD8+ T cell subsets in the blood (A and D), dLN (B and E), and tumors (C and F) of mice carrying B16F10 tumors is shown at specified time points following treatment with either HCW9218 (the first bar shown at each time point in each figure) or HCW9328 (the second bar shown at each time point in each figure). Results show the absolute counts of antigen-experienced (CD44+) CD8+ T cells in (A) blood, (B) dLN, and (C) tumors. DF shows the frequencies of proliferating Ki67+ CD8+ T cells in blood, dLN, and tumors, respectively. Statistical analysis was performed using one-way ANOVA with Graph Pad Prism software. Results using saline are shown as the first single bar in each figure.

[0057] Figure 12 shows the expansion of Tpex and Tex CD8+ T cells in the dLN and tumors of mice after treatment with HCW9328. Flow cytometry staining of Ag-affected subsets in the dLN and tumors of mice carrying B16F10 tumors is shown after treatment with either HCW9328 (second bar shown at each time point in each figure) or HCW9218 (first bar shown at each time point in each figure). Results in (A) show the absolute count of progenitor-depleted (TCF1+PD1+.Tpex) CD8+ T cells in the dLN at the specified time point after treatment in tumor-bearing mice. Results in (B) show Ki67+ proliferating Tpex cells in the dLN. Results in (C) show the absolute count of progenitor-depleted (TCF1+PD1+,Tpex) CD8+ T cells in the tumor at the specified time point after treatment in tumor-bearing mice. Results in (D) show Ki67+ proliferating Tpex cells in the tumor. The results in (E) show the absolute counts of terminally exhausted (TCF1-D1+, Tex) CD8+ T cells in tumors at specified time points after treatment in tumor-bearing mice. Statistical analysis was performed using one-way ANOVA with Graph Pad Prism software. Results using saline are shown as the first single bar in each graph. Detailed Implementation

[0058] This document provides multi-chain chimeric polypeptides comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain comprising: a first sequence at least 80% identical to SEQ ID NO:2, wherein one or both of the following are present: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, glutamine, cysteine, or tyrosine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine, glycine, or valine; and a second sequence at least 80% identical to SEQ ID NO:2, wherein one or both of the following are present: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, glutamine, cysteine, or tyrosine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine, glycine, or valine; (ii) a soluble tissue factor domain; and (iii) a first domain of an affinity domain pair; and (b) The second chimeric polypeptide comprises: (i) a second domain of an affinity domain pair; and (ii) a second target-binding domain comprising: a first sequence that is at least 80% identical to SEQ ID NO:2, wherein one or both of the following are present: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, glutamine, cysteine, or tyrosine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine, glycine, or valine; and a second sequence that is at least 80% identical to SEQ ID NO:2, wherein one or both of the following are present: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, glutamine, cysteine, or tyrosine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine, glycine, or valine; and (ii) the amino acid at position 32 of SEQ ID NO:2 is asparagine, glutamine, cysteine, or tyrosine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine, glycine, or valine; and (ii) the amino acid at position 119 of SEQ ID NO:2 is asparagine, glutamine, cysteine, or tyrosine. The amino acid at position 119 in NO:2 is alanine, glycine, or valine, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated through the binding of the first and second domains of the affinity pair; and the first target-binding domain specifically binds to the ligand of TGF-β receptor II (TGF-βRII), and the second target-binding domain specifically binds to the ligand of TGF-βRII. Various methods for using these multi-chain chimeric polypeptides are also provided herein.

[0059] In some embodiments, the first sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the first sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2.

[0060] In some embodiments, the first sequence of the first target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0061] In some embodiments, the first sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the first sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0062] In some embodiments, the second sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the second sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2.

[0063] In some embodiments, the second sequence of the first target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0064] In some embodiments, the second sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the second sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0065] In some embodiments, the first and second sequences of the first target binding domain are separated by a connector (e.g., any exemplary connector described herein, such as SEQ ID NO:3).

[0066] In some embodiments, the first sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the first sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2.

[0067] In some embodiments, the first sequence of the second target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0068] In some embodiments, the first sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the first sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0069] In some embodiments, the second sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the second sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2.

[0070] In some embodiments, the second sequence of the second target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0071] In some embodiments, the second sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the second sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0072] In some embodiments, the first and second sequences of the first target binding domain are separated by a connector (e.g., any exemplary connector described herein, such as SEQ ID NO:3).

[0073] In a preferred embodiment, the multi-chain chimeric polypeptide comprises (a) a first chimeric polypeptide comprising: (i) a first target-binding domain comprising: (i) a first sequence at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine; and a second sequence at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine; (ii) a soluble tissue factor domain comprising a sequence at least 90% identical to SEQ ID NO:1; and (iii) a first domain of an affinity domain pair comprising a sequence at least 90% identical to SEQ ID NO:13; and (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair comprising a sequence at least 90% identical to SEQ ID NO:2; (ii) a first sequence that is at least 90% identical to SEQ ID NO:2; and (ii) a second target-binding domain comprising: a first sequence that is at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine; and a second sequence that is at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first and second domains of the affinity domain pair.

[0074] As used herein, the term "chimera" refers to a polypeptide comprising amino acid sequences (e.g., domains) originally derived from two different sources (e.g., two different naturally occurring proteins, such as those from the same or different species). For example, a chimeric polypeptide may comprise domains from at least two different naturally occurring human proteins. In some instances, a chimeric polypeptide may comprise a domain (e.g., scFv) as a synthetic sequence and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide may comprise at least two different domains (e.g., two different scFvs) as a synthetic sequence.

[0075] An "antigen-binding domain" is one or more protein domains (e.g., formed from amino acids from a single polypeptide, or from amino acids from two or more polypeptides, e.g., the same or different polypeptides) capable of specifically binding to one or more different antigens. In some instances, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the antigen-binding domain may be an antibody or a fragment thereof. In some embodiments, the antigen-binding domain may contain an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are known in the art.

[0076] "Soluble tissue factor domain" refers to a polypeptide that shares at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with a segment of a wild-type mammalian tissue factor protein (e.g., wild-type human tissue factor protein) lacking both transmembrane and intracellular domains. Non-limiting examples of soluble tissue factor domains are described herein.

[0077] The term "soluble interleukin receptor" is used herein in its broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its native ligands (e.g., under physiological conditions, such as in phosphate-buffered saline at room temperature). For example, a soluble interleukin receptor may contain a sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical) to the extracellular domain of a wild-type interleukin receptor and retains its ability to specifically bind one or more of its native ligands, but lacks its transmembrane domain (and optionally, further lacks its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0078] The term "soluble cytokine receptor" is used herein in its broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its natural ligands (e.g., under physiological conditions, such as in phosphate-buffered saline at room temperature). For example, a soluble cytokine receptor may contain a sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical) to the extracellular domain of a wild-type cytokine receptor and retains its ability to specifically bind one or more of its natural ligands, but lacks its transmembrane domain (and optionally, further lacks its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0079] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. Further examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0080] “Affinity” refers to the sum of the strengths of non-covalent interactions between an antigen-binding site and its binding partner (e.g., an antigen or epitope). Unless otherwise stated, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between a member of the antigen-binding domain and the antigen or epitope. The affinity of molecule X for its partner Y can be expressed using the dissociation equilibrium constant (K0). D The following describes in more detail the kinetic components that contribute to the dissociation equilibrium constant. Affinity can be measured by methods commonly known in the art, including those described herein. For example, affinity can be determined using surface plasmon resonance (SPR) techniques (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Other methods for determining the affinity of an antigen-binding domain for its corresponding antigen or epitope are known in the art.

[0081] As used herein, a “multi-chain polypeptide” refers to a polypeptide comprising two or more (e.g., three, four, five, six, seven, eight, nine, or ten) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), wherein the two or more protein chains are associated by non-covalent bonds to form a quaternary structure.

[0082] The term "affinity domain pair" refers to a pair of domains smaller than 1 × 10⁻⁶. -7M (for example, 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) of K D Affinity domain pairs are two distinct protein domains that bind specifically to each other. In some instances, the affinity domain pair may be a pair of naturally occurring proteins. In some embodiments, the affinity domain pair may be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs are described herein.

[0083] The term "epitope" refers to a portion of an antigen that specifically binds to its antigen-binding domain. An epitope may consist, for example, of surface-accessible amino acid residues and / or sugar side chains, and may possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that binding to the former may be lost in the presence of denaturing solvents, while binding to the latter may not be lost. An epitope may contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding. Methods for identifying epitopes bound to antigen-binding domains are known in the art.

[0084] "Immune effector cells" are cells of the mammalian immune system that are capable of directly or indirectly recognizing pathogenic cells (e.g., cancer cells) in mammals and / or causing cell arrest or cell death in those cells. Non-limiting examples of immune effector cells include macrophages, T lymphocytes (e.g., cytotoxic T lymphocytes and T helper cells), natural killer cells, neutrophils, monocytes, and eosinophils. Other examples of immune effector cells are known in the art.

[0085] The term "treatment" refers to the improvement of at least one symptom of a disorder. In some instances, the disorder being treated is cancer, and the improvement of at least one symptom of the cancer includes reducing the abnormal proliferation of factors, gene expression, signal transduction, translation, and / or secretion. Generally, treatment methods involve administering a therapeutically effective amount of a composition to a subject who needs or has been identified as needing such treatment, the composition reducing at least one symptom of the disorder.

[0086] As used herein, the term “neuroinflammatory” refers to an inflammatory response within the central nervous system (CNS) (e.g., the brain and / or spinal cord) and can be characterized by a large number of cellular and molecular changes within the brain or CNS (e.g., the production of one or more of cytokines, chemokines, reactive oxygen species, and second messengers). In some embodiments, neuroinflammatory can be chronic inflammation caused by toxic metabolites, autoimmunity, aging, microorganisms, viruses, traumatic brain injury, or spinal cord injury.

[0087] As used herein, the term "neuroinflammatory disorder" refers to a condition in which an immune response impairs components of the nervous system, such as the brain, spinal cord, and / or optic nerve. In some embodiments, neuroinflammatory disorders may be associated with aging or traumatic brain injury. For example, neuroinflammatory disorders may include neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, and multiple sclerosis). In some instances, neuroinflammatory disorders may include, but are not limited to, fibromyalgia, Huntington's disease, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), acute disseminated encephalomyelitis (ADEM), acute optic neuritis (AON), transverse myelitis, or neuromyelitis optica (NMO). In some instances, neuroinflammatory disorders may include various mental illnesses (e.g., schizophrenia, autism, depression, and other mood disorders). In some embodiments, increased levels of neuroinflammation can accelerate brain aging and the progression of certain diseases, including sarcopenia.

[0088] The phrase "progression rate of neuroinflammatory disorders" refers to the rate at which a neuroinflammatory disorder begins to worsen or leads to the gradual loss of neurons in the CNS region. For example, a neuroinflammatory disorder can progress at a rapid rate (e.g., days or weeks) or at a slower rate (e.g., months or years). In some embodiments, the progression rate of a neuroinflammatory disorder can be measured by molecular imaging (e.g., positron emission tomography (PET)), monitoring microglial activation or astrocyte responses (e.g., immunohistochemistry, immunocytochemistry), or magnetic resonance methods (e.g., magnetic resonance imaging (MRI) or magnetic resonance spectroscopy (MRS)) or by cognitive assessment.

[0089] In some examples of any multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide may each independently be about 50 amino acids to about 3000 amino acids, about 100 amino acids to about 2000 amino acids, about 200 amino acids to about 1000 amino acids, about 300 amino acids to about 900 amino acids, about 400 amino acids to about 800 amino acids, about 500 amino acids to about 700 amino acids, or about 600 amino acids. An illustration of the exemplary multi-chain chimeric polypeptides provided herein is depicted on... Figure 1 and 2 middle.

[0090] In some embodiments of any multi-chain chimeric peptide described herein, a first target-binding domain (e.g., any first target-binding domain described herein) and a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) are directly adjacent to each other in the first chimeric peptide. In some embodiments of any multi-chain chimeric peptide described herein, the first chimeric peptide further includes 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 soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) in the first chimeric peptide.

[0091] In some embodiments of any multi-chain chimeric polypeptide described herein, a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and the first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) in the first chimeric polypeptide.

[0092] In some embodiments of any multi-chain chimeric peptides described herein, the second domain of the affinity domain pair (e.g., any exemplary second domain of any exemplary affinity domain pair 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 peptide. In some embodiments of any multi-chain chimeric peptides described herein,

[0093] The second chimeric polypeptide further includes a linker sequence (e.g., any exemplary second domain of any exemplary affinity pair described herein) between the second domain of the affinity pair in the second chimeric polypeptide and the second target-binding domain (e.g., any exemplary second target-binding domain described herein).

[0094] The non-limiting aspects of these chimeric peptides, nucleic acids, vectors, cells, and methods are described below and can be used in any combination without limitation. Further aspects of these chimeric peptides, nucleic acids, vectors, cells, and methods are known in the art.

[0095] Organization Factor

[0096] Human tissue factor (TGF) 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 contains two phosphorylation sites at Ser253 and Ser258, and one S-palmitoylation site at Cys245. No deletions or mutations of the cytoplasmic domains were found to affect TGF's coagulation activity. TGF has an S-palmitoylation site at Cys245 in its intracellular domain. Cys245 is located at the amino acid terminus of the intracellular domain and is close to the membrane surface. TGF's transmembrane domain consists of a single α-helix.

[0097] The extracellular domain of tissue factor, composed of two fibronectin type III domains, is linked to the transmembrane domain via a six-amino acid linker. This linker provides conformational flexibility, decoupling the extracellular domain of tissue factor from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module consists of two overlapping β-sheets, with the top fold containing three antiparallel β-chains and the bottom fold containing four β-chains. The β-chains are linked by β-loops between chains βA and βB, βC and βD, and βE and βF, all of which are conformationally conserved in both modules. Three short α-helical segments connect the β-chains. A unique feature of tissue factor is the 17-amino acid β-hairpin between the β10 and β11 chains, which is not a common element in 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 type III fibronectin domain structure is characteristic of the immunoglobulin-like protein folding family and is conserved in a variety of extracellular proteins.

[0098] Once the proenzyme FVII binds to tissue to form the active tissue factor-FVIIa complex, it is rapidly converted to FVIIa through limited proteolysis. FVIIa cycles at a concentration of approximately 0.1 nM (1% of plasma FVII) and can also bind directly to tissue factor. Allosteric interactions between tissue factor and FVIIa on the tissue factor-FVIIa complex significantly increase the enzymatic activity of FVIIa: the hydrolysis rate of small chromophore substrates increases by approximately 20 to 100 times, and the activation rate of natural macromolecular substrates FIX and FX increases by nearly a million times. Consistent with the allosteric activation of the FVIIa active site after tissue factor binding, the formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., after exposure of phosphatidyl-L-serine on the membrane surface) via Ca2+... 2+ The dependent pathway increases the rate of FIX or FX activation by an additional 1,000-fold. Compared to free FVIIa, the overall FX activation of the tissue factor-FVIIa-phospholipid complex increases by approximately one million-fold, a key regulator of the coagulation cascade.

[0099] FVII is a single-chain polypeptide of approximately 50 kDa, composed of 406 amino acid residues, featuring an N-terminal γ-carboxyglutamate-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII communicates via an Ile- molecule in the short linker region between the EGF2 and protease domains. 154 -Arg 152 The specific proteolytic cleavage of the bond activates it to FVIIa. This cleavage leads to the light and heavy chains passing through Cys... 135 and Cys 262 The individual disulfide bonds hold them together. FVIIa is held together by its N-terminal GLA domain via Ca 2+ It binds to the phospholipid membrane in a dependent manner. Immediately adjacent to the C-terminus of the GLA domain are an aromatic stack and two EGF domains. The aromatic stack connects the GLA to the binding of a single Ca²⁺ molecule. 2+ The EGF1 domain of the ion. This Ca 2+ Occupation of the binding site increases the amide degradation activity of FVIIa and tissue factor association. The catalytic triad is composed of His 193 Asp 242 and Ser 344 Composition, and with a single Ca within the FVIIa protease domain 2+ The binding of ions is crucial to its catalytic activity. FVII protein hydrolysis activates it to FVIIa, which is released into Ile. 153 The newly formed amino terminus is folded back and inserted into the activation bag, along with Asp 343The carboxylate forms a salt bridge, thereby creating an oxyanion pore. This salt bridge formation is crucial for FVIIa activity. However, oxyanion pore formation does not occur in free FVIIa after proteolytic activation. Therefore, FVIIa cycles in its enzyme-as-an enzyme state, which is difficult for plasma protease inhibitors to recognize, resulting in a cycling half-life of approximately 90 minutes.

[0100] The tissue factor-mediated localization of the FVIIa active site above the membrane surface is crucial for FVIIa orientation toward homologous substrates. Free FVIIa, when bound to the membrane, employs a stable, extended structure with its active site located approximately 80 Å above the membrane surface. Upon binding with tissue factor, the FVIIa active site relocates to approximately 6 Å closer to the membrane. This modulation may facilitate proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using FVIIa without the GLA domain, the active site has been shown to remain at a similar distance above the membrane, demonstrating that tissue factor fully supports FVIIa active site localization even in the absence of FVIIa-membrane interactions. Further data indicate that tissue factor supports full FVIIa proteolytic activity as long as its extracellular domain is somehow bound to the membrane surface. However, increasing the FVIIa active site above the membrane surface to greater than 80 Å significantly reduces the ability of the tissue factor-FVIIa complex to activate FX, but does not diminish the tissue factor-FVIIa amide degradation activity.

[0101] Alanine scanning mutagenesis has been used to assess the role of specific amino acid side chains in the extracellular domain of tissue factor in interacting 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 sites at which alanine substitution resulted in a decrease in binding affinity for FVIIa to 1 / 10 to 1 / 5. Most of these residue side chains were found to be well exposed to the solvent in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand binding sites were located in a wide region at the boundary between the two modules. In the C-module, the Arg residue located on the protruding BC ring... 135 and Phe 140 Provides independent contact with FVIIa. Leu 133 Located at the bottom of the finger-like structure and filling the crack between the two modules. This is for Lys 20 Thr 60 Asp 58 and Ile 22The major clusters of key binding residues provide continuity. 60 Partial exposure to the solvent may result in a localized structural effect rather than significant contact with the ligand. The binding site extends to the concave side of the inter-module corner, involving Glu. 24 and Gln 110 And possibly even further residues Val 207 The combined region extends from Asp58 to Lys 48 Lys 46 、Gln 37 Asp 44 and Trp 45 On the convex region formed. Trp 45 and Asp 44 The interaction with FVIIa is not independent, indicating that Trp 45 The abrupt change in position may reflect the effect of this side chain on adjacent Asp. 44 and Gln 37 The structural importance of local packaging of the side chains. The interaction region further contains two surface-exposed aromatic residues, Phe 76 and Tyr 78 They form part of the hydrophobic clusters in the N-module.

[0102] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as certain protease-activated receptors. Mutation analysis has identified many residues that, when mutated, support the intact FVIIa amide degradation activity against small peptide substrates but lack its ability to support activation against macromolecular substrates (i.e., FVII, FIX, and FX) (Ruf et al., J Biol Chem 267(31): 22206-22210, 1992; Ruf et al., J Biol Chem 267(9): 6375-6381, 1992; Huang et al., J Biol Chem 271(36): 21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25): 7380-7387, 2000). The tissue factor loop region at residues 159-165, and residues in or adjacent to this flexible loop, have been shown to be crucial for the proteolytic activity of the tissue factor-FVIIa complex. This defines the proposed substrate-binding extra-site region of tissue factor, which is quite far from the FVIIa active site. Replacing glycine residues with slightly larger alanine residues significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility provided by glycine is essential for the 159-165 residue loop for large molecular substrate recognition by tissue factor.

[0103] residue Lys 165 and Lys 166 They have also been shown to be important for substrate recognition and binding. Mutations of any of these residues to alanine result in a significant reduction in tissue factor cofactor function. Lys 165 and Lys 166 They are facing away from each other, with Lys among them. 165 In most tissue factor-FVIIa structures, it points to FVIIa, while Lys 166 Pointing to the substrate-binding site region in the crystal structure. Lys of FVIIa 165 and Gla 35 The hypothetical salt bridge formation between them would support the idea that the interaction between tissue factor and the GLA domain of FVIIa regulates 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 (possibly adjacent to the EGF1 domain), and that the presence of the FVIIa GLA domain may directly or indirectly regulate these interactions.

[0104] Soluble tissue factor domain

[0105] In some embodiments of any peptide, composition, or method described herein, the soluble tissue factor domain may be a wild-type tissue factor peptide lacking a signal sequence, transmembrane domain, and intracellular domain. In some instances, the soluble tissue factor domain may be a tissue factor mutant, wherein the wild-type tissue factor peptide lacks a signal sequence, transmembrane domain, and intracellular domain, and has been further modified at selected amino acids. In some instances, the soluble tissue factor domain may be a soluble human tissue factor domain. In some instances, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some instances, the soluble tissue factor domain may be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, mouse soluble tissue factor domains, rat soluble tissue factor domains, and mutant soluble tissue factor domains are shown below.

[0106] Exemplary soluble human tissue factor domain (SEQ ID NO:1)

[0107] SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0108] An exemplary nucleic acid encoding a soluble human tissue factor domain (SEQ ID NO:9)

[0109] AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

[0110] In some embodiments, the soluble tissue factor domain may contain a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:1. In some embodiments, the soluble tissue factor domain may comprise the sequence of SEQ ID NO:1, wherein one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) are removed from its N-terminus, and / or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) are removed from its C-terminus.

[0111] As understood in the art, those skilled in the art will recognize that conserved amino acid mutations across different mammalian species are more likely to reduce protein activity and / or structural stability, while non-conserved amino acid mutations across different mammalian species are less likely to reduce protein activity and / or structural stability.

[0112] In some examples of any multi-chain chimeric peptides described herein, the soluble tissue factor domain cannot bind factor VIIa. In some examples of any multi-chain chimeric peptides described herein, the soluble tissue factor domain does not convert inactive factor X into factor Xa. In some embodiments of any multi-chain chimeric peptides described herein, the multi-chain chimeric peptide does not stimulate blood clotting in mammals.

[0113] In some instances, the soluble tissue factor domain may be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble rat tissue factor domain.

[0114] In some instances, the soluble tissue factor domain does not contain one or more of the following (e.g., two, three, four, five, six, or seven): lysine at amino acid position 20 of the mature wild-type human tissue factor protein; isoleucine at amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at amino acid position 94 of the mature wild-type human tissue factor protein; arginine at amino acid position 135 of the mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 of the mature wild-type human tissue factor protein.

[0115] In some instances, the soluble tissue factor domain may be encoded by a nucleic acid containing a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:9.

[0116] In some embodiments, the soluble tissue factor domain may have a total length of about 20 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 40 amino acids to about 180 amino acids, about 50 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids, about 70 amino acids to about 120 amino acids, about 80 amino acids to about 110 amino acids, or about 90 amino acids to about 100 amino acids.

[0117] Connector sequence

[0118] In some embodiments, the adapter sequence may be a flexible adapter sequence. Non-limiting examples of adapter sequences that may be used are described in Klein et al., Protein Engineering, Design & Selection 27(10):325–330, 2014; Priyanka et al., Protein Sci. 22(2):153–167, 2013. In some instances, the adapter sequence is a synthetic adapter sequence.

[0119] In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide may comprise one, two, three, four, five, six, seven, eight, nine, or ten adapter sequences (e.g., the same or different adapter sequences, such as any exemplary adapter sequences described herein or known in the art). In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide may comprise one, two, three, four, five, six, seven, eight, nine, or ten adapter sequences (e.g., the same or different adapter sequences, such as any exemplary adapter sequences described herein or known in the art).

[0120] In some embodiments, the adapter sequence may have a total length of 1 amino acid to about 100 amino acids.

[0121] In some embodiments, the linker is enriched with glycine (Gly or G) residues. In some embodiments, the linker is enriched with serine (Ser or S) residues. In some embodiments, the linker is enriched with both glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

[0122] In some embodiments, the adapter sequence may contain or consist of GGGGSGGGGSGGGGS (SEQ ID NO:3). In some embodiments, the adapter sequence may be encoded by a nucleic acid containing or consisting of the following: GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO:14). In some embodiments, the adapter sequence may contain or consist of the following: GGGSGGGS.

[0123] First target binding domain and second target binding domain

[0124] In some embodiments, the first sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the first sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2.

[0125] In some embodiments, the first sequence of the first target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0126] In some embodiments, the first sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the first sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0127] In some embodiments, the second sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the second sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2.

[0128] In some embodiments, the second sequence of the first target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0129] In some embodiments, the second sequence of the first target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the second sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0130] In some embodiments, the first and second sequences of the first target binding domain are separated by a connector (e.g., any exemplary connector described herein, such as SEQ ID NO:3).

[0131] In some embodiments, the first sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO: 2. In some embodiments, the first sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO: 2.

[0132] In some embodiments, the first sequence of the second target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0133] In some embodiments, the first sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the first sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0134] In some embodiments, the second sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the second sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2.

[0135] In some embodiments, the second sequence of the second target-binding domain comprises alanine, glycine, or valine at amino acid position 119 of SEQ ID NO:2.

[0136] In some embodiments, the second sequence of the second target-binding domain comprises asparagine, glutamine, cysteine, or tyrosine at amino acid position 32 of SEQ ID NO:2 and alanine, glycine, or valine at amino acid position 119. In some embodiments, the second sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0137] In some embodiments, the first and second sequences of the first target binding domain are separated by a connector (e.g., any exemplary connector described herein, such as SEQ ID NO:3).

[0138] Exemplary first or second sequence (SEQ ID NO:2)

[0139] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPD

[0140] In some embodiments, the first sequence and / or the second sequence of the first target-binding domain and / or the second target-binding domain comprises a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:2.

[0141] In some embodiments, the first sequence and / or the second sequence of the first target-binding domain and / or the second target-binding domain are encoded by nucleic acids comprising sequences that are at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identical to any of SEQ ID NO: 23, 24, 25, and 26.

[0142] An exemplary nucleic acid (SEQ ID NO:23) encoding an exemplary first or second sequence.

[0143] ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCTGAAGTTTCCCCAGCTCTGCAAGTTCTGC AACGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAG ACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCTCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGAC GCC TGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT

[0144] An exemplary nucleic acid encoding an exemplary first or second sequence (SEQ ID NO:24)

[0145] ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGC AAT GTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAA ACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGAC GCT TGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

[0146] An exemplary nucleic acid encoding an exemplary first or second sequence (SEQ ID NO: 25)

[0147] ATCCCACCTCACGTGCAGAAAAGCGTCAATAATGACATGATCGTGACTGACAATAACGGCGCCGTCAAGTTTCCACAGCTGTGTAAGTTCTGC AAC GTGAGATTTTCCACATGCGACAACCAGAAGAGCTGTATGAGCAACTGCAGCATCACAAGCATCTGCGAGAAGCCACAAGAGGTCTGCGTGGCCGTCTGGAGAAAGAACGACGAGAACATCACTCTCGAGACTGTGTGTCACGACCCTAAGCTCCCATACCATGACTTCATCCTCGAGGATGCTGCCTCCCCTAAATGCATTATGAAGGAGAAAAAAAAGCCCGGCGAGACATTCTTTATGTGCAGCTGCTCCTCCGAC GCC TGCAACGACAACATCATCTTTAGCGAAGAATACAACACTAGCAACCCAGAT

[0148] Exemplary nucleic acid encoding an exemplary first or second sequence (SEQ ID NO: 26)

[0149] ATCCCACCACACGTGCAGAAGTCCGTCAACAACGACATGATTGTGACTGACAACAACGGCGCCGTGAAGTTCCCACAACTCTGCAAGTTTTGC AAT GTGAGGTTCTCCACATGTGACAACCAGAAAAGCTGCATGTCCAACTGCTCCATCACTAGCATCTGTGAGAAACCTCAAGAGGTCTGTGTGGCTGTGTGGAGGAAGAACGATGAGAACATCACACTCGAGACAGTCTGCCACGACCCAAAGCTGCCATATCACGACTTCATTCTCGAGGACGCCGCCAGCCCTAAGTGCATCATGAAGGAGAAGAAGAAGCCCGGCGAGACATTTTTCATGTGTAGCTGCAGCTCCGAT GCC TGTAACGACAATATTATCTTTAGCGAGGAGTATAACACATCCAATCCAGAC

[0150] In some embodiments, the first target-binding domain and / or the second target-binding domain comprises a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:4.

[0151] Exemplary first target-binding domain and / or sequence target-binding domain (SEQ ID NO:4)

[0152] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDGGGGSGG GGSGGGGSIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPD

[0153] In some embodiments, the first target-binding domain and / or the second target-binding domain are encoded by nucleic acids comprising sequences that are at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:27 or SEQ ID NO:28.

[0154] An exemplary sequence encoding an exemplary first target-binding domain and / or a second target-binding domain (SEQ ID NO: 27).

[0155] ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCTGAAGTTTCCCCAGCTCTGCAAGTTCTGC AACGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGAC GCC TGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGC AAT GTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGAC GCT TGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

[0156] Exemplary sequences encoding an exemplary first target binding domain and / or a second target binding domain (SEQ ID NO:28)

[0157] ATCCCACCTCACGTGCAGAAAAGCGTCAATAATGACATGATCGTGACTGACAATAACGGCGCCGTCAAGTTTCCACAGCTGTGTAAGTTCTGC AACGTGAGATTTTCCACATGCGACAACCAGAAGAGCTGTATGAGCAACTGCAGCATCACAAGCATCTGCGAGAAGCCACAAGAGGTCTGCGTGGCCGTCTGGAGAAAGAACGACGAGAACATCACTCTCGAG ACTGTGTGTCACGACCCTAAGCTCCCATACCATGACTTCATCCTCGAGGATGCTTGCCTCCCCTAAATGCATTATGAAGGAGAAAAAAAAGCCCGGCGAGACATTCTTTATGTGCAGCTGCTCCTCCGAC GCC TGCAACGACAACATCATCTTTAGCGAAGAATACAACACTAGCAACCCAGATGGCGGCGGAGGATCCGGAGGAGGAGGCTCCGGAGGCGGAGGCAGCATCCCACCACACGTGCAGAAGTCCGTCAACAACGACATGATTGTGACTGACAACAACGGCCGCCGTGAAGTTCCCACAACTCTGCAAGTTTTGC AAT GTGAGGTTCTCCACATGTGACAACCAGAAAAGCTGCATGTCCAACTGCTCCATCACTAGCATCTGTGAGAAACCTCAAGAGGTCTGTGTGGCTGTGTGGAGGAAGAACGATGAGAACATCACACTCGAG ACAGTCTGCCACGACCCAAAGCTGCCATATCACGACTTCATTCTCGAGGACGCCGCCAGCCCTAAGTGCATCATGAAGGAGAAGAAGAAGCCCGGCGAGACATTTTTCATGTGTAGCTGCAGCTCCGAT GCC TGTAACGACAATATTATCTTTAGCGAGGAGTATAACACATCCAATCCAGAC

[0158] Any target-binding domain described herein can be dissociated with the following equilibrium constant (K) D Binding of ligands to TGF-βRII: less than 1 × 10 -7 M, less than 1 × 10 -8 M, less than 1 × 10 -9 M, less than 1 × 10 -10 M, less than 1 × 10 -11M, less than 1 × 10 -12 M, or less than 1 × 10 -13 M. In some embodiments, the antigen-binding protein constructs provided herein can be configured as follows K D Binding to the identification antigen: approximately 1 × 10 -3 M to approximately 1 × 10 -5 M, approximately 1 × 10 -4 M to approximately 1 × 10 -6 M, approximately 1 × 10 -5 M to approximately 1 × 10 -7 M, approximately 1 × 10 -6 M to approximately 1 × 10 -8 M, approximately 1 × 10 -7 M to approximately 1 × 10 -9 M, approximately 1 × 10 -8 M to approximately 1 × 10 -10 M, or approximately 1 × 10 -9 M to approximately 1 × 10 -11 M (including end values).

[0159] Any target-binding domain described herein can be in the range of about 1 pM to 30 nM (e.g., about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about 650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about 350 pM, about 1 pM to about 30 ... pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM, about 2 pM to about 30 nM, about 2 pM to about 25 nM, about 2 pM to about 20 nM, about 2 pM to about 15 nM, about 2 pM to about 10 nM, about 2 pM to about 5 nM, about 2 pM to about 2 nM, about 2 pM to about 1 nM, approximately 2 pM to approximately 950 pM, approximately 2 pM to approximately 900 pM, approximately 2 pM to approximately 850 pM, approximately 2 pM to approximately 800 pM, approximately 2 pM to approximately 750 pM, approximately 2 pM to approximately 700 pM, approximately 2 pM to approximately 650 pM, approximately 2 pM to approximately 600 pM, approximately 2 pM to approximately 550 pM, approximately 2 pM to approximately 500 pM, approximately 2 pM to approximately 450 pM, approximately 2 pM to approximately 400 pM, approximately 2 pM to approximately 350 pM, approximately 2 pM to approximately 300 pM, approximately 2 pM to approximately 250 pM, approximately 2 pM to approximately 200 pM, approximately 2 pM to approximately 150 pM, approximately 2 pM to approximately 100 pM, approximately 2 pM to approximately 90 pM, approximately 2 pM to approximately 80 pM, approximately 2 pM to approximately 70 pM, approximately 2 pM to approximately 60 pM, approximately 2 pM to about 50 pM, about 2 pM to about 40 pM, about 2 pM to about 30 pM, about 2 pM to about 20 pM, about 2 pM to about 10 pMpM, approximately 2 pM to approximately 5 pM, approximately 2 pM to approximately 4 pM, approximately 2 pM to approximately 3 pM, approximately 5 pM to approximately 30 nM, approximately 5 pM to approximately 25 nM, approximately 5 pM to approximately 20 nM, approximately 5 pM to approximately 15 nM, approximately 5 pM to approximately 10 nM, approximately 5 pM to approximately 5 nM, approximately 5 pM to approximately 2 nM, approximately 5 pM to approximately 1 nM, approximately 5 pM to approximately 950 pM, approximately 5 pM to approximately 900 pM, approximately 5 pM to approximately 850 pM, approximately 5 pM to approximately 800 pM, approximately 5 pM to approximately 750 pM, approximately 5 pM to approximately 700 pM, approximately 5 pM to approximately 650 pM, approximately 5 pM to approximately 600 pM, approximately 5 pM to approximately 550 pM, approximately 5 pM to approximately 500 pM, approximately 5 pM to approximately 450 pM, approximately 5 pM to approximately 400 pM, approximately 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM to about 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30 pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM to about 30 nM, about 10 pM to about 25 nM, about 10 pM to about 20 nM, about 10 pM to about 15 nM, about 10 pM to about 10 nM, about 10 pM to about 5 nM, about 10 pM to about 2 nM, about 10 pM to about 1 nM, about 10 pM to about 950 pM, about 10 pM to about 900 pM, about 10 pM to about 850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about 10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about 600 pM, about 10 pM to about 550 pM, about 10 pM to about 500 pM, about 10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about 350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about 10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 100 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM pM, approximately 10 pM to approximately 60 pM, approximately 10 pM to approximately 50 pM, approximately 10 pM to approximately 40 pM, approximately 10 pM to approximately 30 pM, approximately 10 pM to approximately 20 pM, approximately 15pM to about 30 nM, about 15 pM to about 25 nM, about 15 pM to about 20 nM, about 15 pM to about 15 nM, about 15 pM to about 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about 15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about 900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 pM, about 15 pM to about 750 pM, about 15 pM to about 700 pM, about 15 pM to about 650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about 15 pM to about 500 pM, about 15 pM to about 450 pM, about 15 pM to about 400 pM, about 15 pM to about 350 pM, about 15 pM to about 300 pM, about 15 pM to about 250 pM, about 15 pM to about 200 pM, about 15 pM to about 150 pM, about 15 pM to about 100 pM, about 15 pM to about 90 pM, about 15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about 60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15 pM to about 30 pM, about 15 pM to about 20 pM, about 20 pM to about 30 nM, about 20 pM to about 25 nM, about 20 pM to about 20 nM, about 20 pM to about 15 nM, about 20 pM to about 10 nM, about 20 pM to about 5 nM, about 20 pM to about 2 nM, about 20 pM to about 1 nM, about 20 pM to about 950 pM, about 20 pM to approximately 900 pM, approximately 20 pM to approximately 850 pM, approximately 20 pM to approximately 800 pM, approximately 20 pM to approximately 750 pM, approximately 20 pM to approximately 700 pM, approximately 20 pM to approximately 650 pM, approximately 20 pM to approximately 600 pM, approximately 20 pM to approximately 550 pM, approximately 20 pM to approximately 500 pM, approximately 20 pM to approximately 450 pM, approximately 20 pM to approximately 400 pM, approximately 20 pM to approximately 350 pM, approximately 20 pM to approximately 300 pM, approximately 20 pM to approximately 250 pM, approximately 20 pM to approximately 20 pM, approximately 200 pM to approximately 150 pM, approximately 20 pM to approximately 100 pM, approximately 20 pM to approximately 90 pM, approximately 20 pM to approximately 80 pM, approximately 20 pM to approximately 70 pM, approximately 20 pM to approximately 60 pM pM, approximately 20 pM to approximately 50 pM, approximately 20 pM to approximately 40 pM, approximately 20 pM to approximately 30 pM, approximately 30 pM to approximately 30 nM, approximately 30 pM to approximately 25 nM, approximately 30 pM to approximately 30 nMnM, approximately 30 pM to approximately 15 nM, approximately 30 pM to approximately 10 nM, approximately 30 pM to approximately 5 nM, approximately 30 pM to approximately 2 nM, approximately 30 pM to approximately 1 nM, approximately 30 pM to approximately 950 pM, approximately 30 pM to approximately 900 pM, approximately 30 pM to approximately 850 pM, approximately 30 pM to approximately 800 pM, approximately 30 pM to approximately 750 pM, approximately 30 pM to approximately 700 pM, approximately 30 pM to approximately 650 pM, approximately 30 pM to approximately 600 pM, approximately 30 pM to approximately 550 pM, approximately 30 pM to approximately 500 pM, approximately 30 pM to approximately 450 pM, approximately 30 pM to approximately 400 pM, approximately 30 pM to approximately 350 pM, approximately 30 pM to approximately 300 pM, approximately 30 pM to approximately 250 pM, approximately 30 pM to approximately 200 pM pM, approximately 30 pM to approximately 150 pM, approximately 30 pM to approximately 100 pM, approximately 30 pM to approximately 90 pM, approximately 30 pM to approximately 80 pM, approximately 30 pM to approximately 70 pM, approximately 30 pM to approximately 60 pM, approximately 30 pM to approximately 50 pM, approximately 30 pM to approximately 40 pM, approximately 40 pM to approximately 30 nM, approximately 40 pM to approximately 25 nM, approximately 40 pM to approximately 30 nM, approximately 40 pM to approximately 15 nM, approximately 40 pM to approximately 10 nM, approximately 40 pM to approximately 5 nM, approximately 40 pM to approximately 2 nM, approximately 40 pM to approximately 1 nM, approximately 40 pM to approximately 950 pM, approximately 40 pM to approximately 900 pM, approximately 40 pM to approximately 850 pM, approximately 40 pM to approximately 800 pM, approximately 40 pM to approximately 750 pM, approximately 40 pM to approximately 700 pM, approximately 40 pM to approximately 650 pM, approximately 40 pM to approximately 600 pM, approximately 40 pM to approximately 550 pM, approximately 40 pM to approximately 500 pM, approximately 40 pM to approximately 450 pM, approximately 40 pM to approximately 400 pM, approximately 40 pM to approximately 350 pM, approximately 40 pM to approximately 300 pM, approximately 40 pM to approximately 250 pM, approximately 40 pM to approximately 200 pM, approximately 40 pM to approximately 150 pM, approximately 40 pM to approximately 100 pM, approximately 40 pM to approximately 90 pM, approximately 40 pM to approximately 80 pM, approximately 40 pM to approximately 70 pM, approximately 40 pM to approximately 60 pM, approximately 40 pM to approximately 50 pM, approximately 50 pM to approximately 30 nM, approximately 50 pM to approximately 25 nM, approximately 50 pM to approximately 30 nM, approximately 50 pM to approximately 15 nM, approximately 50 pM to approximately 10 nM, approximately 50 pM to approximately 5 nM, approximately 50 pM to approximately 2 nM, approximately 50 pM to approximately 1 nM, approximately 50 pM to approximately 950 pM, approximately 50pM to approximately 900 pM, approximately 50 pM to approximately 850 pM, approximately 50 pM to approximately 800 pM, approximately 50 pM to approximately 750 pM, approximately 50 pM to approximately 700 pM, approximately 50 pM to approximately 650 pM, approximately 50 pM to approximately 600 pM, approximately 50 pM to approximately 550 pM, approximately 50 pM to approximately 500 pM, approximately 50 pM to approximately 450 pM, approximately 50 pM to approximately 400 pM, approximately 50 pM to approximately 350 pM, approximately 50 pM to approximately 300 pM, approximately 50 pM to approximately 250 pM, approximately 50 pM to approximately 200 pM, approximately 50 pM to approximately 150 pM, approximately 50 pM to approximately 100 pM, approximately 50 pM to approximately 90 pM, approximately 50 pM to approximately 80 pM, approximately 50 pM to approximately 70 pM, approximately 50 pM to approximately 60 pM, approximately 60 pM to approximately 30 nM, approximately 60 pM to approximately 25 nM, approximately 60 pM to approximately 30 nM, approximately 60 pM to approximately 15 nM, approximately 60 pM to approximately 10 nM, approximately 60 pM to approximately 5 nM, approximately 60 pM to approximately 2 nM, approximately 60 pM to approximately 1 nM, approximately 60 pM to approximately 950 pM, approximately 60 pM to approximately 900 pM, approximately 60 pM to approximately 850 pM, approximately 60 pM to approximately 800 pM, approximately 60 pM to approximately 750 pM, approximately 60 pM to approximately 700 pM, approximately 60 pM to approximately 650 pM, approximately 60 pM to approximately 600 pM, approximately 60 pM to approximately 550 pM, approximately 60 pM to approximately 500 pM, approximately 60 pM to approximately 450 pM, approximately 60 pM to approximately 400 pM, approximately 60 pM to approximately 350 pM pM, approximately 60 pM to approximately 300 pM, approximately 60 pM to approximately 250 pM, approximately 60 pM to approximately 200 pM, approximately 60 pM to approximately 150 pM, approximately 60 pM to approximately 100 pM, approximately 60 pM to approximately 90 pM, approximately 60 pM to approximately 80 pM, approximately 60 pM to approximately 70 pM, approximately 70 pM to approximately 30 nM, approximately 70 pM to approximately 25 nM, approximately 70 pM to approximately 30 nM, approximately 70 pM to approximately 15 nM, approximately 70 pM to approximately 10 nM, approximately 70 pM to approximately 5 nM, approximately 70 pM to approximately 2 nM, approximately 70 pM to approximately 1 nM, approximately 70 pM to approximately 950 pM, approximately 70 pM to approximately 900 pM, approximately 70 pM to approximately 850 pM, approximately 70 pM to approximately 800 pM, approximately 70 pM to approximately 750 pM, approximately 70 pM to approximately 700 pM, approximately 70 pM to approximately 650 pM, approximately 70 pM to approximately 600 pM, approximately 70 pM to approximately 550 pM, approximately 70 pM to approximately 500 pM, approximately 70 pM to approximately 450 pM, approximately 70pM to approximately 400 pM, approximately 70 pM to approximately 350 pM, approximately 70 pM to approximately 300 pM, approximately 70 pM to approximately 250 pM, approximately 70 pM to approximately 200 pM, approximately 70 pM to approximately 150 pM, approximately 70 pM to approximately 100 pM, approximately 70 pM to approximately 90 pM, approximately 70 pM to approximately 80 pM, approximately 80 pM to approximately 30 nM, approximately 80 pM to approximately 25 nM, approximately 80 pM to approximately 30 nM, approximately 80 pM to approximately 15 nM, approximately 80 pM to approximately 10 nM, approximately 80 pM to approximately 5 nM, approximately 80 pM to approximately 2 nM, approximately 80 pM to approximately 1 nM, approximately 80 pM to approximately 950 pM, approximately 80 pM to approximately 900 pM, approximately 80 pM to approximately 850 pM, approximately 80 pM to approximately 800 pM, approximately 80 pM to approximately 750 pM pM, approximately 80 pM to approximately 700 pM, approximately 80 pM to approximately 650 pM, approximately 80 pM to approximately 600 pM, approximately 80 pM to approximately 550 pM, approximately 80 pM to approximately 500 pM, approximately 80 pM to approximately 450 pM, approximately 80 pM to approximately 400 pM, approximately 80 pM to approximately 350 pM, approximately 80 pM to approximately 300 pM, approximately 80 pM to approximately 250 pM, approximately 80 pM to approximately 200 pM, approximately 80 pM to approximately 150 pM, approximately 80 pM to approximately 100 pM, approximately 80 pM to approximately 90 pM, approximately 90 pM to approximately 30 nM, approximately 90 pM to approximately 25 nM, approximately 90 pM to approximately 30 nM, approximately 90 pM to approximately 15 nM, approximately 90 pM to approximately 10 nM, approximately 90 pM to approximately 5 nM, approximately 90 pM to approximately 2 nM, approximately 90 pM to approximately 1 nM, approximately 90 pM to approximately 950 pM, approximately 90 pM to approximately 900 pM, approximately 90 pM to approximately 850 pM, approximately 90 pM to approximately 800 pM, approximately 90 pM to approximately 750 pM, approximately 90 pM to approximately 700 pM, approximately 90 pM to approximately 650 pM, approximately 90 pM to approximately 600 pM, approximately 90 pM to approximately 550 pM, approximately 90 pM to approximately 500 pM, approximately 90 pM to approximately 450 pM, approximately 90 pM to approximately 400 pM, approximately 90 pM to approximately 350 pM, approximately 90 pM to approximately 300 pM, approximately 90 pM to approximately 250 pM, approximately 90 pM to approximately 200 pM, approximately 90 pM to approximately 150 pM, approximately 90 pM to approximately 100 pM, approximately 100 pM to approximately 30 nM, approximately 100 pM to approximately 25 nM, approximately 100 pM to approximately 30 nM, approximately 100 pM to approximately 15 nM, approximately 100 pM to approximately 10 nM, approximately 100 pM to approximately 5 nM, approximately 100 pM to approximately 2nM, approximately 100 pM to approximately 1 nM, approximately 100 pM to approximately 950 pM, approximately 100 pM to approximately 900 pM, approximately 100 pM to approximately 850 pM, approximately 100 pM to approximately 800 pM, approximately 100 pM to approximately 750 pM, approximately 100 pM to approximately 700 pM, approximately 100 pM to approximately 650 pM, approximately 100 pM to approximately 600 pM, approximately 100 pM to approximately 550 pM, approximately 100 pM to approximately 500 pM, approximately 100 pM to approximately 450 pM, approximately 100 pM to approximately 400 pM, approximately 100 pM to approximately 350 pM, approximately 100 pM to approximately 300 pM, approximately 100 pM to approximately 250 pM, approximately 100 pM to approximately 200 pM, approximately 100 pM to approximately 150 pM, approximately 150 pM to approximately 300 pM nM, approximately 150 pM to approximately 25 nM, approximately 150 pM to approximately 30 nM, approximately 150 pM to approximately 15 nM, approximately 150 pM to approximately 10 nM, approximately 150 pM to approximately 5 nM, approximately 150 pM to approximately 2 nM, approximately 150 pM to approximately 1 nM, approximately 150 pM to approximately 950 pM, approximately 150 pM to approximately 900 pM, approximately 150 pM to approximately 850 pM, approximately 150 pM to approximately 800 pM, approximately 150 pM to approximately 750 pM, approximately 150 pM to approximately 700 pM, approximately 150 pM to approximately 650 pM, approximately 150 pM to approximately 600 pM, approximately 150 pM to approximately 550 pM, approximately 150 pM to approximately 500 pM, approximately 150 pM to approximately 450 pM, approximately 150 pM to approximately 400 pM, approximately 150 pM to approximately 350 pM, approximately 150 pM to approximately 300 pM, approximately 150 pM to approximately 250 pM, approximately 150 pM to approximately 200 pM, approximately 200 pM to approximately 30 nM, approximately 200 pM to approximately 25 nM, approximately 200 pM to approximately 30 nM, approximately 200 pM to approximately 15 nM, approximately 200 pM to approximately 10 nM, approximately 200 pM to approximately 5 nM, approximately 200 pM to approximately 2 nM, approximately 200 pM to approximately 1 nM, approximately 200 pM to approximately 950 pM, approximately 200 pM to approximately 900 pM, approximately 200 pM to approximately 850 pM, approximately 200 pM to approximately 800 pM, approximately 200 pM to approximately 750 pM, approximately 200 pM to approximately 700 pM, approximately 200 pM to approximately 650 pM, approximately 200 pM to approximately 600 pM pM, approximately 200 pM to approximately 550 pM, approximately 200 pM to approximately 500 pM, approximately 200 pM to approximately 450 pM, approximately 200 pM to approximately 400 pM, approximately 200 pM to approximately 350 pM, approximately 200 pM to approximately 300 pMpM, approximately 200 pM to approximately 250 pM, approximately 300 pM to approximately 30 nM, approximately 300 pM to approximately 25 nM, approximately 300 pM to approximately 30 nM, approximately 300 pM to approximately 15 nM, approximately 300 pM to approximately 10 nM, approximately 300 pM to approximately 5 nM, approximately 300 pM to approximately 2 nM, approximately 300 pM to approximately 1 nM, approximately 300 pM to approximately 950 pM, approximately 300 pM to approximately 900 pM, approximately 300 pM to approximately 850 pM, approximately 300 pM to approximately 800 pM, approximately 300 pM to approximately 750 pM, approximately 300 pM to approximately 700 pM, approximately 300 pM to approximately 650 pM, approximately 300 pM to approximately 600 pM, approximately 300 pM to approximately 550 pM, approximately 300 pM to approximately 500 pM, approximately 300 pM to approximately 450 pM, approximately 300 pM to approximately 400 pM, approximately 300 pM to approximately 350 pM, approximately 400 pM to approximately 30 nM, approximately 400 pM to approximately 25 nM, approximately 400 pM to approximately 30 nM, approximately 400 pM to approximately 15 nM, approximately 400 pM to approximately 10 nM, approximately 400 pM to approximately 5 nM, approximately 400 pM to approximately 2 nM, approximately 400 pM to approximately 1 nM, approximately 400 pM to approximately 950 pM, approximately 400 pM to approximately 900 pM, approximately 400 pM to approximately 850 pM, approximately 400 pM to approximately 800 pM, approximately 400 pM to approximately 750 pM, approximately 400 pM to approximately 700 pM, approximately 400 pM to approximately 650 pM, approximately 400 pM to approximately 600 pM, approximately 400 pM to approximately 550 pM pM, approximately 400 pM to approximately 500 pM, approximately 500 pM to approximately 30 nM, approximately 500 pM to approximately 25 nM, approximately 500 pM to approximately 30 nM, approximately 500 pM to approximately 15 nM, approximately 500 pM to approximately 10 nM, approximately 500 pM to approximately 5 nM, approximately 500 pM to approximately 2 nM, approximately 500 pM to approximately 1 nM, approximately 500 pM to approximately 950 pM, approximately 500 pM to approximately 900 pM, approximately 500 pM to approximately 850 pM, approximately 500 pM to approximately 800 pM, approximately 500 pM to approximately 750 pM, approximately 500 pM to approximately 700 pM, approximately 500 pM to approximately 650 pM, approximately 500 pM to approximately 600 pM, approximately 500 pM to approximately 550 pM, approximately 600 pM to approximately 30 nM, approximately 600 pM to approximately 25 nM, approximately 600 pM to approximately 30 nM, approximately 600 pM to approximately 15 nM, approximately 600 pM to approximately 10 nM, approximately 600 pM to approximately 5 nM, approximately 600 pM to approximately 2 nM, approximately 600 pM to approximately 1 nM, approximately 600pM to approximately 950 pM, approximately 600 pM to approximately 900 pM, approximately 600 pM to approximately 850 pM, approximately 600 pM to approximately 800 pM, approximately 600 pM to approximately 750 pM, approximately 600 pM to approximately 700 pM, approximately 600 pM to approximately 650 pM, approximately 700 pM to approximately 30 nM, approximately 700 pM to approximately 25 nM, approximately 700 pM to approximately 30 nM, approximately 700 pM to approximately 15 nM, approximately 700 pM to approximately 10 nM, approximately 700 pM to approximately 5 nM, approximately 700 pM to approximately 2 nM, approximately 700 pM to approximately 1 nM, approximately 700 pM to approximately 950 pM, approximately 700 pM to approximately 900 pM, approximately 700 pM to approximately 850 pM, approximately 700 pM to approximately 800 pM, approximately 700 pM to approximately 750 pM pM, approximately 800 pM to approximately 30 nM, approximately 800 pM to approximately 25 nM, approximately 800 pM to approximately 30 nM, approximately 800 pM to approximately 15 nM, approximately 800 pM to approximately 10 nM, approximately 800 pM to approximately 5 nM, approximately 800 pM to approximately 2 nM, approximately 800 pM to approximately 1 nM, approximately 800 pM to approximately 950 pM, approximately 800 pM to approximately 900 pM, approximately 800 pM to approximately 850 pM, approximately 900 pM to approximately 30 nM, approximately 900 pM to approximately 25 nM, approximately 900 pM to approximately 30 nM, approximately 900 pM to approximately 15 nM, approximately 900 pM to approximately 10 nM, approximately 900 pM to approximately 5 nM, approximately 900 pM to approximately 2 nM, approximately 900 pM to approximately 1 nM, approximately 900 pM to approximately 950 pM, approximately 1 nM to about 30 nM, about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30 nM, about 10 nM to about 25 nM nM, approximately 10 nM to approximately 20 nM, approximately 10 nM to approximately 15 nM, approximately 15 nM to approximately 30 nM, approximately 15 nM to approximately 25 nM, approximately 15 nM to approximately 20 nM, approximately 20 nM to approximately 30 nMK between nM and approximately 20 nM to approximately 25 nM) D It binds to ligands of TGF-βRII (e.g., TGF-β).

[0160] Any target-binding domain described herein may be in the range of about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about 6 nM, about 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nM to about 10 ... K between nM to 9 nM, about 4 nM to 8 nM, about 4 nM to 7 nM, about 4 nM to 6 nM, about 4 nM to 5 nM, about 5 nM to 10 nM, about 5 nM to 9 nM, about 5 nM to 8 nM, about 5 nM to 7 nM, about 5 nM to 6 nM, about 6 nM to 10 nM, about 6 nM to 9 nM, about 6 nM to 8 nM, about 6 nM to 7 nM, about 7 nM to 10 nM, about 7 nM to 9 nM, about 7 nM to 8 nM, about 8 nM to 10 nM, about 8 nM to 9 nM and about 9 nM to 10 nM) D It binds to the ligand of TGFβRII.

[0161] K0 of any antigen-binding protein construct described herein can be determined using a variety of different methods known in the art, such as electrophoretic mobility variation assays, membrane filtration binding assays, surface plasmon resonance assays, and biomolecular binding kinetic assays. D value.

[0162] In some embodiments of any multi-chain chimeric polypeptide described herein, the soluble TGF-β receptor is either a soluble TGF-β receptor II (TGF-β RII) (see, for example, those described in Yung et al., Am. J. Resp. Crit. Care Med. [American Journal of Respiratory and Critical Care Medicine] 194(9):1140-1151, 2016) or a soluble TGF-β RIII (see, for example, those described in Heng et al., Placenta [Placement] 57:320, 2017).

[0163] Other target-binding domains

[0164] In some embodiments of any multi-chain chimeric polypeptide, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art), wherein at least one of the one or more additional antigen-binding domains is located between a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide may further include a linker sequence (e.g., any exemplary soluble tissue factor domain described herein) between at least one of a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein or known in the art) and one or more other target-binding domains (e.g., any exemplary target-binding domain described herein or known in the art), and / or a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of one or more other target-binding domains (e.g., any exemplary target-binding domain described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein).

[0165] In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains located at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to a first domain of an affinity domain pair in the first chimeric polypeptide (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of the one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to the first target-binding domain of the first chimeric polypeptide (e.g., any exemplary target-binding domain described herein or known in the art). In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domain described herein or known in the art) and the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art).

[0166] In some embodiments of any multi-chain chimeric peptides described herein, at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is disposed at the N-terminus and / or C-terminus of the first chimeric peptide, and at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is located between a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) disposed at the N-terminus is directly adjacent to the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) or the first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any linker sequence described herein or known in the art) disposed between at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) and the first target-binding domain (e.g., any exemplary first domain of any exemplary affinity pair described herein). In some embodiments, at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) disposed at the C-terminus is directly adjacent to the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) or the first domain of the affinity pair (e.g., any exemplary first domain of any exemplary affinity pair described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary target-binding domain described herein or known in the art) disposed between at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) and a first target-binding domain (e.g., any exemplary first domain described herein of any exemplary affinity domain pair) or an affinity domain pair (e.g., any exemplary first domain described herein of any exemplary affinity domain pair).In some embodiments, at least one of one or more additional target-binding domains (e.g., any exemplary soluble tissue factor domain described herein) located between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and the first domain of the affinity domain pair (e.g., any first domain described herein or any exemplary affinity domain pair described herein) is directly adjacent to the first domain of the soluble tissue factor domain and / or the first domain of the affinity domain pair. In some embodiments, the first chimeric polypeptide further comprises a linker sequence disposed between: (i) at least one of a soluble tissue factor domain (e.g., any exemplary soluble tissue factor described herein or known in the art) and one or more additional target-binding domains (e.g., any exemplary target-binding domain described herein or known in the art), the one or more additional target-binding domains being located between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein), and / or (ii) between the first domain of an affinity domain pair and at least one of one or more additional target-binding domains, the one or more additional target-binding domains being located between the soluble tissue factor domain and the first domain of the affinity domain pair.

[0167] In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) at the N-terminus and / or C-terminus of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to a second domain of an affinity pair in the second chimeric polypeptide (e.g., any exemplary second domain of any exemplary affinity pair described herein). In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of the one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) and a second domain of an affinity pair (e.g., any second domain of any exemplary affinity pair described herein). In some embodiments, at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to a second target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) in the second chimeric polypeptide and a second target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art).

[0168] In some embodiments of any multi-chain chimeric polypeptide described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) first target-binding domains, second target-binding domains, and one or more additional target-binding domains specifically bind to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) first target-binding domains, second target-binding domains, and one or more additional target-binding domains specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) first target-binding domains, second target-binding domains, and 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 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 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 one or more additional target-binding domains each contain the same amino acid sequence.

[0169] In some embodiments of any multi-chain chimeric peptide described herein, a first target-binding domain, a second target-binding domain, and one or more additional target-binding domains specifically bind to different antigens. In some embodiments of any multi-chain chimeric peptide described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and one or more target-binding domains are antigen-binding domains. In some embodiments, the first target-binding domain, the second target-binding domain, and one or more additional target-binding domains are each an antigen-binding domain (e.g., scFv or a single-domain antibody).

[0170] Affinity domain pairs

[0171] In some embodiments, the multi-chain chimeric polypeptide comprises: 1) a first chimeric polypeptide comprising a first domain of an affinity domain pair, and 2) a second chimeric polypeptide comprising a second domain of an affinity domain pair, such that the first and second chimeric polypeptides associate through the binding of the first and second domains of the affinity domain pair. In some embodiments, the affinity domain pair is the sushi domain from the α chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. The sushi domain, also known as a short concordant repeat or type 1 glycoprotein motif, is a common motif in protein-protein interactions. Sushi domains have been found on many protein-binding molecules, including complement components C1r, C1s, factor H, and C2m, as well as non-immune molecules factor XIII and β2-glycoprotein. A typical Sushi domain has approximately 60 amino acid residues and includes four cysteine ​​residues (Ranganathan, Pac. Symp Biocomput. [Pacific Biocomputation Symposium] 2000:155-67). The first cysteine ​​can form a disulfide bond with the third cysteine, and the second cysteine ​​can form a disulfide bridge with the fourth cysteine. In some embodiments where one member of the affinity domain pair is soluble IL-15, soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments where one member of the affinity domain pair is the α chain of the human IL-15 receptor (IL15Rα), human IL15Rα is the mature, full-length IL15Rα. In some embodiments, the affinity domain pair is barnase and barnstar. In some embodiments, the affinity domain pair is PKA and AKAP. In some embodiments, the affinity domain pair is an adaptor / docking tag module based on a mutated RNase I fragment (Rossi, Proc Natl Acad Sci USA. [Proceedings of the National Academy of Sciences] 103:6841-6846, 2006; Sharkey et al., Cancer Res. [Cancer Research] 68:5282-5290, 2008; Rossi et al., Trends Pharmacol Sci. [Trends in Pharmaceutical Sciences] 33:474-481, 2012) or a SNARE module based on the interaction of protein synaptic proteins, synaptic binding proteins, synaptic vesicle proteins, and SNAP25 (Deyev et al., Nat Biotechnol. [Nature Biotechnology] 1486-1492, 2003).

[0172] In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide comprises a first domain of an affinity domain pair, and a second chimeric polypeptide of a multi-chain chimeric polypeptide comprises a second domain of an affinity domain pair, wherein the first domain of the affinity domain pair and the second domain of the affinity domain pair are in an affinity ratio of less than 1 × 10⁻⁶. -7 M, less than 1 × 10 -8 M, less than 1 × 10 -9 M, less than 1 × 10 -10 M, less than 1 × 10 -11 M, less than 1 × 10 -12 M, or less than 1 × 10 -13 The dissociation equilibrium constant of M (K) D They are combined with each other. In some embodiments, the first domain of the affinity domain pair and the second domain of the affinity domain pair are about 1 × 10 -4 M to approximately 1 × 10 -6 M, approximately 1 × 10 -5 M to approximately 1 × 10 -7 M, approximately 1 × 10 -6 M to approximately 1 × 10 -8 M, approximately 1 × 10 -7 M to approximately 1 × 10 -9 M, approximately 1 × 10 -8 M to approximately 1 × 10 -10 M, approximately 1 × 10 -9 M to approximately 1 × 10 -11 M, approximately 1 × 10 -10 M to approximately 1 × 10 -12 M, approximately 1 × 10 -11 M to approximately 1 × 10 -13 M, approximately 1 × 10 -4 M to approximately 1 × 10 -5 M, approximately 1 × 10 -5 M to approximately 1 × 10 -6 M, approximately 1 × 10 -6 M to approximately 1 × 10 -7 M, approximately 1 × 10 -7 M to approximately 1 × 10 -8 M, approximately 1 × 10 -8 M to approximately 1 × 10 -9 M, approximately 1 × 10 -9 M to approximately 1 × 10 -10 M, approximately 1 × 10 -10 M to approximately 1 × 10-11 M, approximately 1 × 10 -11 M to approximately 1 × 10 -12 M or approximately 1 × 10 -12 M to approximately 1 × 10 -13 M (including end values) of K D They combine with each other. Any of the various methods known in the art (e.g., electrophoretic mobility variation measurement, membrane filtration binding measurement, surface plasmon resonance, and biomolecular binding kinetics measurement, etc.) can be used to determine the K-value of the binding between the first domain and the second domain of the affinity domain pair. D value.

[0173] In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first domain of an affinity domain pair, and a second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a second domain of an affinity domain pair, wherein the length of the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both is about 10 to 100 amino acids. For example, the length of the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both can be about 10 to 100 amino acids, about 15 to 100 amino acids, about 20 to 100 amino acids, about 25 to 100 amino acids, about 30 to 100 amino acids, about 35 to 100 amino acids, about 40 to 100 amino acids, about 45 to 100 amino acids, about 50 to 100 amino acids, or about 55 to 100 amino acids. 00 amino acid length, approximately 60 to 100 amino acid length, approximately 65 to 100 amino acid length, approximately 70 to 100 amino acid length, approximately 75 to 100 amino acid length, approximately 80 to 100 amino acid length, approximately 85 to 100 amino acid length, approximately 90 to 100 amino acid length, approximately 95 to 100 amino acid length, approximately 10 to 95 amino acid length, approximately 10 to 85 amino acid length, approximately 10 to 80 amino acids The sequence of amino acids is as follows: approximately 10 to 75 amino acids, approximately 10 to 70 amino acids, approximately 10 to 65 amino acids, approximately 10 to 60 amino acids, approximately 10 to 55 amino acids, approximately 10 to 50 amino acids, approximately 10 to 45 amino acids, approximately 10 to 40 amino acids, approximately 10 to 35 amino acids, approximately 10 to 30 amino acids, approximately 10 to 25 amino acids, approximately 10 to 20 amino acids, approximately 10 to 15 amino acids. The length of the amino acid is approximately 20 to 30 amino acids, approximately 30 to 40 amino acids, approximately 40 to 50 amino acids, approximately 50 to 60 amino acids, approximately 60 to 70 amino acids, approximately 70 to 80 amino acids, approximately 80 to 90 amino acids, approximately 90 to 100 amino acids, approximately 20 to 90 amino acids, approximately 30 to 80 amino acids, approximately 40 to 70 amino acids, approximately 50 to 60 amino acids, or any range between these lengths. In some embodiments, the length of the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0174] In some embodiments, either the first and / or second domains of the affinity domain pair disclosed herein may contain one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at their N-terminus and / or C-terminus, provided that the function of the first and / or second domains of the affinity domain pair remains intact. For example, the sushi domain of the α chain (IL15Rα) of the human IL-15 receptor may contain one or more additional amino acids at its N-terminus and / or C-terminus while still retaining its ability to bind to soluble IL-15. Alternatively or additionally, soluble IL-15 may contain one or more additional amino acids at its N-terminus and / or C-terminus while still retaining its ability to bind to the sushi domain of the α chain (IL15Rα) of the human IL-15 receptor.

[0175] Non-limiting instances of the sushi domain of the α chain from the IL-15 receptor α (IL15Rα) may contain sequences that are 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 the following sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO:11). In some embodiments, the sushi domain of the α chain from IL15Rα may be encoded by a nucleic acid comprising the following: ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO:12).

[0176] In some embodiments, soluble IL-15 may comprise a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical: NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:13). In some embodiments, soluble IL-15 may be encoded by a nucleic acid comprising the following sequence: AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO:14).

[0177] In some embodiments, soluble IL-15 may contain a D8N amino acid substitution. In some embodiments, soluble IL-15 with the D8N mutant (IL15D8N) may contain 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 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to the following sequence: NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:33). In some embodiments, soluble IL-15 with the D8N mutant (IL15D8N) may be encoded by a nucleic acid containing the following sequence:

[0178] AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAG TATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT (SEQ ID NO:34).

[0179] signal sequence

[0180] In some embodiments, the multi-chain chimeric polypeptide comprises a first chimeric polypeptide containing a signal sequence at its N-terminus. In some embodiments, the multi-chain chimeric polypeptide comprises a second chimeric polypeptide containing a signal sequence at its N-terminus. In some embodiments, both the first and second chimeric polypeptides of the multi-chain chimeric polypeptide contain a signal sequence. Those skilled in the art will understand that a signal sequence is an amino acid sequence present at the N-terminus of many endogenously produced proteins that guides the protein into secretory pathways (e.g., guiding the protein to reside in certain intracellular organelles, reside in the cell membrane, or be secreted from the cell). Signal sequences are heterogeneous and their primary amino acid sequences vary considerably. However, signal sequences are typically 16 to 30 amino acids in length and contain a hydrophilic, generally positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region containing a signal peptidase cleavage site.

[0181] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a signal sequence having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO:15). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a signal sequence encoded by the following nucleic acid sequence: ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO:16), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO:17), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO:18).

[0182] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 19). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a signal sequence having the amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO: 20). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a signal sequence having the amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 21). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a signal sequence having the amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO:22). Those skilled in the art will recognize other suitable signal sequences that can be used in the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide described herein.

[0183] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a signal sequence of about 10 to 100 amino acids in length. For example, the signal sequence may be about 10 to 100 amino acids long, about 15 to 100 amino acids long, about 20 to 100 amino acids long, about 25 to 100 amino acids long, about 30 to 100 amino acids long, about 35 to 100 amino acids long, about 40 to 100 amino acids long, about 45 to 100 amino acids long, about 50 to 100 amino acids long, about 55 to 100 amino acids long, or about 60 to 100 amino acids long. The amino acid length is approximately 65 to 100 amino acids, approximately 70 to 100 amino acids, approximately 75 to 100 amino acids, approximately 80 to 100 amino acids, approximately 85 to 100 amino acids, approximately 90 to 100 amino acids, approximately 95 to 100 amino acids, approximately 10 to 95 amino acids, approximately 10 to 85 amino acids, approximately 10 to 80 amino acids, approximately 10 to 75 amino acids. The acid length is approximately 10 to 70 amino acids long, approximately 10 to 65 amino acids long, approximately 10 to 60 amino acids long, approximately 10 to 55 amino acids long, approximately 10 to 50 amino acids long, approximately 10 to 45 amino acids long, approximately 10 to 40 amino acids long, approximately 10 to 35 amino acids long, approximately 10 to 30 amino acids long, approximately 10 to 25 amino acids long, approximately 10 to 20 amino acids long, approximately 10 to 15 amino acids long, approximately 20 The length of the signal sequence can be up to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range between these. In some embodiments, the length of the signal sequence is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0184] In some embodiments, any signal sequence disclosed herein may contain one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10 or more amino acids) at its N-terminus and / or C-terminus, provided that the function of the signal sequence remains intact. For example, a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO:19) may contain one or more additional amino acids at its N-terminus or C-terminus while still retaining the ability to guide the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide into the secretory pathway.

[0185] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide contain a signal sequence that directs the multi-chain chimeric polypeptide into the extracellular space. Such embodiments can be used to produce multi-chain chimeric polypeptides that are relatively easy to isolate and / or purify.

[0186] peptide tags

[0187] In some embodiments, the multi-chain chimeric polypeptide comprises a first chimeric polypeptide containing a peptide tag (e.g., at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, the multi-chain chimeric polypeptide comprises a second chimeric polypeptide containing a peptide tag (e.g., at the N-terminus or C-terminus of the second chimeric polypeptide). In some embodiments, both the first chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide contain peptide tags. In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide contain two or more peptide tags.

[0188] Exemplary peptide tags that may be included in a first chimeric peptide, a second chimeric peptide, or both of a multi-chain chimeric polypeptide include, but are not limited to, AviTag, calmodulin tag, polyglutamic acid tag, E tag, FLAG tag, HA tag, peptides derived from hemagglutinin, his tag, myc tag, NE tag, S tag, SBP tag, Softag 1, Softag 3, Spot tag, Strep tag, TC tag, Ty tag, V5 tag, VSV tag, and Xpress tag. In some embodiments, tissue factor protein is a peptide tag.

[0189] Peptide tags, which can be included in the first chimeric polypeptide, the second chimeric polypeptide, or both of a multi-chain chimeric polypeptide, can be used in any of the wide variety of applications related to multi-chain chimeric polypeptides. For example, peptide tags can be used for the purification of multi-chain chimeric polypeptides. As a non-limiting example, the first chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both of a multi-chain chimeric polypeptide can contain a myc tag; multi-chain chimeric polypeptides containing a first chimeric polypeptide with a myc tag, a second chimeric polypeptide with a myc tag, or both can be purified using an antibody that recognizes the myc tag. A non-limiting example of an antibody that recognizes the myc tag is 9E10, which is available from a non-commercial developmental research hybridoma library. As another non-limiting example, the first chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both of the multi-chain chimeric polypeptide may contain a histidine tag; multi-chain chimeric polypeptides containing a histidine-tagged first chimeric polypeptide, a histidine-tagged second chimeric polypeptide, or both can be purified using nickel or cobalt chelates. Those skilled in the art will understand other suitable tags for purifying multi-chain chimeric polypeptides and agents for binding these tags. In some embodiments, the peptide tag is removed from the first chimeric polypeptide and / or the second chimeric polypeptide after purification. In some embodiments, the peptide tag is not removed from the first chimeric polypeptide and / or the second chimeric polypeptide after purification.

[0190] Peptide tags that may be included in a first chimeric polypeptide, a second chimeric polypeptide, or both of a multi-chain chimeric polypeptide can be used, for example, for immunoprecipitation of the multi-chain chimeric polypeptide, imaging of the multi-chain chimeric polypeptide (e.g., via Western blotting, ELISA, flow cytometry, and / or immunocytochemistry), and / or dissolution of the multi-chain chimeric polypeptide.

[0191] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide comprise a peptide tag of about 10 to 100 amino acids in length. For example, the peptide tag can be about 10 to 100 amino acids long, about 15 to 100 amino acids long, about 20 to 100 amino acids long, about 25 to 100 amino acids long, about 30 to 100 amino acids long, about 35 to 100 amino acids long, about 40 to 100 amino acids long, about 45 to 100 amino acids long, about 50 to 100 amino acids long, about 55 to 100 amino acids long, or about 60 to 100 amino acids long. Long, approximately 65 to 100 amino acids, approximately 70 to 100 amino acids, approximately 75 to 100 amino acids, approximately 80 to 100 amino acids, approximately 85 to 100 amino acids, approximately 90 to 100 amino acids, approximately 95 to 100 amino acids, approximately 10 to 95 amino acids, approximately 10 to 85 amino acids, approximately 10 to 80 amino acids, approximately 10 to 75 amino acids The acid length is approximately 10 to 70 amino acids long, approximately 10 to 65 amino acids long, approximately 10 to 60 amino acids long, approximately 10 to 55 amino acids long, approximately 10 to 50 amino acids long, approximately 10 to 45 amino acids long, approximately 10 to 40 amino acids long, approximately 10 to 35 amino acids long, approximately 10 to 30 amino acids long, approximately 10 to 25 amino acids long, approximately 10 to 20 amino acids long, approximately 10 to 15 amino acids long, approximately 20 The length of the peptide tag can be up to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range between these. In some embodiments, the length of the peptide tag is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0192] The peptide tag contained in the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide can have any suitable length. For example, the length of the peptide tag can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids. In embodiments where the multi-chain chimeric polypeptide contains two or more peptide tags, the two or more peptide tags can have the same or different lengths. In some embodiments, any peptide tag disclosed herein may contain one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminus and / or C-terminus, as long as the peptide tag's function remains intact. For example, the myc tag may contain one or more additional amino acids (e.g., at the N-terminus and / or C-terminus of the peptide tag) while still retaining the ability to be bound by an antibody.

[0193] Exemplary multi-chain chimeric peptide

[0194] The exemplary multi-chain chimeric polypeptides provided herein comprise: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain comprising: a first sequence at least 80% identical to SEQ ID NO:2, wherein one or both of the following are present: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine; and a second sequence at least 80% identical to SEQ ID NO:2, wherein one or both of the following are present: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine; (ii) a soluble tissue factor domain; and (iii) a first domain of an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target-binding domain comprising: a first sequence at least 80% identical to SEQ ID NO:2, wherein one or both of the following are present: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine; (ii) a soluble tissue factor domain; and (iii) a first domain of an affinity domain pair; The first sequence is at least 80% identical to SEQ ID NO:2, wherein one or both of the following conditions are met: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine; and the second sequence is at least 80% identical to SEQ ID NO:2, wherein one or both of the following conditions are met: (A) the amino acid at position 32 of SEQ ID NO:2 is asparagine, and (B) the amino acid at position 119 of SEQ ID NO:2 is alanine.

[0195] The first chimeric polypeptide and the second chimeric polypeptide are associated through the binding of the first and second domains of the affinity pair; and the first target-binding domain specifically binds to the ligand of TGF-β receptor II (TGF-βRII), and the second target-binding domain specifically binds to the ligand of TGF-βRII.

[0196] In some embodiments, the first sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the first sequence of the first target-binding domain comprises alanine at amino acid position 119 of SEQ ID NO:2. In some embodiments, the first sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0197] In some embodiments, the second sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the second sequence of the first target-binding domain comprises alanine at amino acid position 119 of SEQ ID NO:2. In some embodiments, the second sequence of the first target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0198] In some embodiments, the first sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the first sequence of the second target-binding domain comprises alanine at amino acid position 119 of SEQ ID NO:2. In some embodiments, the first sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0199] In some embodiments, the second sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2. In some embodiments, the second sequence of the second target-binding domain comprises alanine at amino acid position 119 of SEQ ID NO:2. In some embodiments, the second sequence of the second target-binding domain comprises asparagine at amino acid position 32 of SEQ ID NO:2 and alanine at amino acid position 119.

[0200] In some embodiments, the first target-binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

[0201] In some embodiments, in the first chimeric polypeptide, the soluble tissue factor domain and the first domain of the affinity domain pair are directly adjacent to each other. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide.

[0202] In some embodiments, in the second chimeric polypeptide, the second domain of the affinity pair is directly adjacent to the second target-binding domain. In some embodiments, the second chimeric polypeptide further includes a linker sequence between the second domain of the affinity pair and the second target-binding domain in the second chimeric polypeptide.

[0203] In some embodiments, the first chimeric polypeptide comprises a sequence that is at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:6 or 7.

[0204] In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:5 or 8.

[0205] Exemplary first chimeric polypeptide (SEQ ID NO:6)

[0206] IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0207] Exemplary first chimeric polypeptide (SEQ ID NO:7)

[0208] MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0209] Exemplary second chimeric polypeptide (SEQ ID NO:5)

[0210] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFP QLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0211] Exemplary second chimeric polypeptide (SEQ ID NO:8)

[0212] MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVT DNNGAVKFPQLCKFCNVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDACNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0213] In some embodiments, the first chimeric polypeptide is encoded by a nucleic acid comprising a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:29 or 30.

[0214] In some embodiments, the second chimeric polypeptide is encoded by a nucleic acid comprising a sequence that is at least 80% identical, at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:31 or 32.

[0215] An exemplary nucleic acid encoding an exemplary first chimeric polypeptide (SEQ ID NO:29)

[0216]

[0217] An exemplary nucleic acid encoding an exemplary first chimeric polypeptide (SEQ ID NO:30)

[0218]

[0219] An exemplary nucleic acid encoding an exemplary second chimeric polypeptide (SEQ ID NO:31)

[0220]

[0221] Exemplary nucleic acid encoding an exemplary second chimeric polypeptide (SEQ ID NO:32)

[0222] ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCCATCCCACCTCACGTGCAGAAAAGCGTCAATAATGACATGATCGTGACTGACAATAACGGCGCCGTCAAGTTTCCACAGCTGTGTAAGTTCTGC AAC GTGAGATTTTCCACATGCGACAACCAGAAGAGCTGTATGAGCAACTGCAGCATCACAAGCATCTGCGAGAAGCCACAAGAGGTCTGCGTGGCCGTCTGGAGAAAGAACGACGAGAACATCACTCTCGAGACTGTGTGTCACGACCCTAAGCTCCCATACCATGACTTCATCCTCGAGGATGCTGCCTCCCCTAAATGCATTATGAAGGAGAAAAAAAAGCCCGGCGAGACATTCTTTATGTGCAGCTGCTCCTCCGAC GCC TGCAACGACAACATCATCTTTAGCGAAGAATACAACACTAGCAACCCAGATGGCGGCGGAGGATCCGGAGGAGGAGGCTCCGGAGGCGGAGGCAGCATCCCACCACACGTGCAGAAGTCCGTCAACAACGACATGATTGTGACTGACAACAACGGCGCCGTGAAGTTCCCACAACTCTGCAAGTTTTGC AAT GTGAGGTTCTCCACATGTGACAACCAGAAAAGCTGCATGTCCAACTGCTCCATCACTAGCATCTGTGAGAAACCTCAAGAGGTCTGTGTGGCTGTGTGGAGGAAGAACGATGAGAACATCACACTCGAGACAGTCTGCCACGACCCAAAGCTGCCATATCACGACTTCATTCTCGAGGACGCCGCCAGCCCTAAGTGCATCATGAAGGAGAAGAAGAAGCCCGGCGAGACATTTTTCATGTGTAGCTGCAGCTCCGAT GCCTGTAACGACAATATTATCTTTAGCGAGGAGTATAACACATCCAATCCAGACATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACAACCCTCTTTAAAGTGCATCCGG

[0223] Composition / Reagent Kit

[0224] This document also provides compositions comprising at least one of the following (e.g., pharmaceutical compositions): any multi-chain chimeric polypeptide described herein, any cell, or any nucleic acid. In some embodiments, the composition comprises at least one of any multi-chain chimeric polypeptides described herein. In some embodiments, the composition comprises any immune cell (e.g., any immune cell described herein, such as any immune cell generated using any of the methods described herein).

[0225] In some embodiments, the pharmaceutical composition is formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical composition may contain a pharmaceutically acceptable carrier (e.g., phosphate-buffered saline).

[0226] The pharmaceutical composition may be administered to subjects in need, either as a single or multiple doses, depending on, for example, the subject's need and tolerance for the dosage and frequency. The formulation should provide an adequate amount of the active agent to effectively treat, prevent, or improve the condition, disease, or symptom.

[0227] This document also provides kits comprising any of the multi-chain chimeric peptides, compositions, nucleic acids, or cells (e.g., immune cells) described herein. In some embodiments, the kit may include instructions for performing any of the methods described herein. In some embodiments, the kit may include at least one dose of any of the pharmaceutical compositions described herein.

[0228] Nucleic acid / vector

[0229] This document also provides nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first nucleic acid may encode a first chimeric polypeptide and a second nucleic acid may encode a second chimeric polypeptide. In some embodiments, a single nucleic acid may encode both the first chimeric polypeptide and the second chimeric polypeptide.

[0230] This document also provides vectors comprising any nucleic acid encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first vector may comprise a nucleic acid encoding a first chimeric polypeptide, and a second vector may comprise a nucleic acid encoding a second chimeric polypeptide. In some embodiments, a single vector may comprise a first nucleic acid encoding a first chimeric polypeptide and a second nucleic acid encoding a second chimeric polypeptide.

[0231] Any vector described herein can be an expression vector. For example, an expression vector may contain a promoter sequence operatively linked to a sequence encoding a first chimeric polypeptide and a second chimeric polypeptide.

[0232] Non-limiting examples of vectors include plasmids, transposons, granules, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector) and any Gateway® vector. Vectors may, for example, contain sufficient cis-acting elements for expression; other elements for expression may be provided by host mammalian cells or in an in vitro expression system. Those skilled in the art will be able to select suitable vectors and mammalian cells to prepare any of the multi-chain chimeric polypeptides described herein.

[0233] cell

[0234] This document also provides cells (e.g., any exemplary cells described herein or known in the art) that contain any nucleic acid described herein encoding any multi-chain chimeric polypeptide described herein (e.g., both a first chimeric polypeptide and a second chimeric polypeptide). This document also provides cells (e.g., any exemplary cells described herein or known in the art) that contain any nucleic acid described herein encoding any first chimeric polypeptide described herein. Furthermore, this document provides cells (e.g., any exemplary cells described herein or known in the art) that contain any nucleic acid described herein encoding any second chimeric polypeptide described herein.

[0235] This document also provides cells (e.g., any exemplary cells described herein or known in the art) that contain any vector described herein encoding any multi-chain chimeric polypeptide described herein (e.g., both a first chimeric polypeptide and a second chimeric polypeptide). This document also provides cells (e.g., any exemplary cells described herein or known in the art) that contain any vector described herein encoding any first chimeric polypeptide described herein. This document also provides cells (e.g., any exemplary cells described herein or known in the art) that contain any vector described herein encoding any second chimeric polypeptide described herein.

[0236] In some embodiments of any of the methods described herein, the cell may be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell having a distinctive membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human), insect, fungal, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell, such as *Saccharomyces cerevisiae*. In some embodiments, the eukaryotic cell is a higher eukaryote, such as a mammalian, avian, plant, or insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary cells and human embryonic kidney cells (e.g., HEK293 cells).

[0237] Methods for introducing nucleic acids and expression vectors into cells (e.g., eukaryotic cells) are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipid transfection, transfection, electroporation, microinjection, calcium phosphate transfection, dendritic macromolecule-based transfection, cationic polymer transfection, cell extrusion, acoustic pore effect, optical transfection, puncture transfection, hydrodynamic delivery, magnetic transfection, viral transduction (e.g., adenovirus and lentivirus transduction), and nanoparticle transfection.

[0238] Methods for generating multi-chain chimeric peptides

[0239] This document also provides methods for generating any of the multi-chain chimeric polypeptides described herein, including: culturing any of the cells described herein in a culture medium under conditions sufficient to induce the generation of the multi-chain chimeric polypeptide; and recovering the multi-chain chimeric polypeptide from the cells and / or the culture medium.

[0240] This document also provides methods for generating any of the multi-chain chimeric peptides described herein, including: culturing any of the cells described herein in a first culture medium under conditions sufficient to induce the generation of a first chimeric peptide; recovering the first chimeric peptide from the cells and / or the first culture medium; culturing any of the cells described herein in a second culture medium under conditions sufficient to induce the generation of a second chimeric peptide; recovering the second chimeric peptide from the cells and / or the second culture medium; and combining (e.g., mixing) the recovered first chimeric peptide and the recovered second chimeric peptide to form a multi-chain chimeric peptide (e.g., any of the multi-chain chimeric peptides described herein).

[0241] The recovery of multi-chain chimeric peptides, first chimeric peptides, or second chimeric peptides from cells (e.g., eukaryotic cells) can be performed using techniques well known in the art (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation, ion exchange chromatography (anion or cation), hydrophobic interaction-based chromatography, metal affinity chromatography, ligand affinity chromatography, and size exclusion chromatography).

[0242] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions favorable to proliferation, differentiation, and growth. In short, cells can be cultured by contacting them (e.g., any cell) with a cell culture medium containing the necessary growth factors and supplements to support cell viability and growth.

[0243] This document also provides multi-chain chimeric polypeptides (e.g., any multi-chain chimeric polypeptides described herein), first chimeric polypeptides (e.g., any first chimeric polypeptide), or second chimeric polypeptides (e.g., any second chimeric polypeptide described herein) generated by any of the methods described herein.

[0244] Methods of stimulating immune cells

[0245] This document also provides methods for stimulating immune cells (e.g., any exemplary immune cells described herein or known in the art), methods comprising contacting the immune cells with an effective amount of any multi-chain chimeric polypeptide described herein or any composition described herein (e.g., a pharmaceutical composition). In some instances, the immune cells are contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to induce immune cell stimulation).

[0246] In some instances, the immune cells have been previously obtained from the subject (e.g., a mammal, such as a human). Some embodiments of these methods further include obtaining the immune cells from the subject prior to the contact step.

[0247] In some instances, immune cells are brought into contact in vivo. In such embodiments, the multi-chain chimeric polypeptide is administered to the subject in an amount sufficient to stimulate immune cells in the subject (e.g., a mammal, such as a human).

[0248] In some instances of any of the methods described herein, immune cells may be immature thymocytes, peripheral blood lymphocytes, primary T cells, pluripotent Th cell precursors, lymphoid progenitor cells, Treg cells, memory T cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8+ cells, etc. + T cells, CD4 + T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, or natural killer cells, or combinations thereof. In some embodiments, the T cells may be progenitor-depleted CD8 progenitor cells. + (Tpex) cells (e.g., CD3) + CD8 + TCF1 + TOX + PD1 +TIGIT + CD27 + Cellular and effector memory CD8 + T (Tem) cells (e.g., CD3) + CD8 + CD44 (high CD62L, low cellularity), central memory CD8 + T (Tcm) cells (e.g., CD3) + CD8 + CD44 (high CD62L high cellularity), virtual memory CD8 + T (Tvm) cells (e.g., CD3) + CD8 + CD44 high CD49d low cellularity), antigen-experienced CD8 + T cells (e.g., CD44) + CCR7 + CD127 + CD122 + CD27 + CD62L + perforin + CD103 + CD69 + Cellular cells, terminally exhausted CD8 + T (Tex) cells (e.g., TOX) + TCF1 - Granulase B + PD-1 + Tim-3 + CD101 + CXCR3 + CCR5 + (cells) or memory stem CD8 + T (Tscm) cells (e.g., CD3) + CD8 + CD45RA, CCR7 + CD27 + CD95 + CXCR3 + cell).

[0249] In some instances, the immune cells have been previously genetically modified to express chimeric antigen receptors or recombinant T-cell receptors. In some instances, the immune cells (e.g., any immune cells described herein) have been previously genetically modified to express co-stimulatory molecules (e.g., CD28).

[0250] Some embodiments of these methods may further include, after the contact step, introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T-cell receptor into an immune cell (e.g., any immune cell described herein). Some embodiments of these methods may further include, after the contact step, introducing a nucleic acid encoding a co-stimulatory molecule (e.g., CD28) into an immune cell (e.g., any immune cell described herein).

[0251] Some embodiments of these methods may further include administering a therapeutically effective amount of immune cells to a subject in need (e.g., any of the exemplary subjects described herein).

[0252] In some instances, participants may be those who have been identified or diagnosed with an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

[0253] In some instances, participants may be those who have been identified or diagnosed with cancer. Non-limiting examples of cancer include: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal 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.

[0254] In some instances, the subject may be a subject who has been diagnosed or identified with an infectious disease. Non-limiting examples of infectious diseases include infections with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus and hepatitis C virus, human papillomavirus, or influenza virus.

[0255] Activation of immune cells can be determined using methods known in the art. For example, activation can be determined by detecting the levels of cytokines and chemokines secreted or upregulated cytotoxic granules and regulatory molecules following activation. Non-limiting examples of cytokines, chemokines, cytotoxic granules, and regulatory molecules secreted or upregulated following activation of immune cells include: IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFα, granzyme, perforin, TGFβ, STAT3, STAT4, STAT5, RORKT, FOXP3, STAT6, and GATA3. These cytokines, chemokines, cytotoxic granules, or regulatory molecules can be detected using immunoassays (e.g., enzyme-linked immunosorbent assays) and quantitative PCR. For example, activation of immune cells can lead to the activation of one or more of any of the cytokines, chemokines, cytotoxic granules, or regulatory molecules described herein (e.g., any IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFα, granzymes, etc.). The percentage of perforin, TGFβ, STAT3, STAT4, STAT5, RORKT, FOXP3, and GATA3 increases by about 1% to about 800% (e.g., about 1% to about 750%, about 1% to about 700%, about 1% to about 650%, about 1% to about 600%, about 1% to about 550%, about 1% to about 500%, about 1% to about 450%, about 1% to about 400%, about 1% to about 350%, about 1% to about 30%). 0%, about 1% to about 280%, about 1% to about 260%, about 1% to about 240%, about 1% to about 220%, about 1% to about 200%, about 1% to about 180%, about 1% to about 160%, about 1% to about 140%, about 1% to about 120%, about 1% to about 100%, about 1% to about 90%, about 1% to about 80%, about 1% to about 70%, about 1% to about 60%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40% %, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 800%, about 5% to about 750%, about 5% to about 700%, about 5% to about 650%, about 5% to about 600%, about 5% to about 550%, about 5% to about 500%, about 5% to about 450%, about 5% to about 400%, about 5% to about 350%.About 5% to about 300%, about 5% to about 280%, about 5% to about 260%, about 5% to about 240%, about 5% to about 220%, about 5% to about 200%, about 5% to about 180%, about 5% to about 160%, about 5% to about 140%, about 5% to about 120%, about 5% to about 100%, about 5% to about 90%, about 5% to about 80%, about 5% to about 70%, about 5% to about 60%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 800%, about 10% to about 750%. About 10% to about 700%, about 10% to about 650%, about 10% to about 600%, about 10% to about 550%, about 10% to about 500%, about 10% to about 450%, about 10% to about 400%, about 10% to about 350%, about 10% to about 300%, about 10% to about 280%, about 10% to about 260%, about 10% to about 240%, about 10% to about 220%, about 10% to about 200%, about 10% to about 180%, about 10% to about 160%, about 10% to about 140%, about 10% to about 120%, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%. Approximately 10% to approximately 50%, approximately 10% to approximately 45%, approximately 10% to approximately 40%, approximately 10% to approximately 35%, approximately 10% to approximately 30%, approximately 10% to approximately 25%, approximately 10% to approximately 20%, approximately 10% to approximately 15%, approximately 15% to approximately 800%, approximately 15% to approximately 750%, approximately 15% to approximately 700%, approximately 15% to approximately 650%, approximately 15% to approximately 600%, approximately 15% to approximately 550%, approximately 15% to approximately 500%, approximately 15% to approximately 450%, approximately 15% to approximately 400%, approximately 15% to approximately 350%, approximately 15% to approximately 300%, approximately 15% to approximately 280%, approximately 15% to approximately 260%, approximately 15% to approximately 240%, approximately 15% to approximately 220%, approximately 15% About 200%, about 15% to about 180%, about 15% to about 160%, about 15% to about 140%, about 15% to about 120%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 15% to about 20%, about 20% to about 800%, about 20% to about 750%, about 20% to about 700%, about 20% to about 650%, about 20% to about 600%, about 20% to about 550%, about 20% to about 500%.About 20% to about 450%, about 20% to about 400%, about 20% to about 350%, about 20% to about 300%, about 20% to about 280%, about 20% to about 260%, about 20% to about 240%, about 20% to about 220%, about 20% to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 20% to Approximately 25%, approximately 25% to approximately 800%, approximately 25% to approximately 750%, approximately 25% to approximately 700%, approximately 25% to approximately 650%, approximately 25% to approximately 600%, approximately 25% to approximately 550%, approximately 25% to approximately 500%, approximately 25% to approximately 450%, approximately 25% to approximately 400%, approximately 25% to approximately 350%, approximately 25% to approximately 300%, approximately 25% to approximately 280%, approximately 25% to approximately 260%, approximately 25% to approximately 240%, approximately 25% to approximately 220%, approximately 25% to approximately 200%, approximately 25% to approximately 180%, approximately 25% to approximately 160%, approximately 25% to approximately 140%, approximately 25% to approximately 120%, approximately 25% to approximately 100%, approximately 25% to approximately 90%, approximately 25% About 80%, about 25% to about 70%, about 25% to about 60%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, about 25% to about 35%, about 35% to about 800%, about 35% to about 750%, about 35% to about 700%, about 35% to about 650%, about 35% to about 600%, about 35% to about 550%, about 35% to about 500%, about 35% to about 450%, about 35% to about 400%, about 35% to about 350%, about 35% to about 300%, about 35% to about 280%, about 35% to about 260%, about 35% to about 240%, about 35% to about 220%, about 35% to about 200%, about 35% to about 1 80%, about 35% to about 160%, about 35% to about 140%, about 35% to about 120%, about 35% to about 100%, about 35% to about 90%, about 35% to about 80%, about 35% to about 70%, about 35% to about 60%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 40% to about 800%, about 40% to about 750%, about 40% to about 700%, about 40% to about 650%, about 40% to about 600%, about 40% to about 550%, about 40% to about 500%, about 40% to about 450%, about 40% to about 400%, about 40% to about 350%, about 40% to about 300%, about 40% to about 280%.Approximately 40% to approximately 260%, approximately 40% to approximately 240%, approximately 40% to approximately 220%, approximately 40% to approximately 200%, approximately 40% to approximately 180%, approximately 40% to approximately 160%, approximately 40% to approximately 140%, approximately 40% to approximately 120%, approximately 40% to approximately 100%, approximately 40% to approximately 90%, approximately 40% to approximately 80%, approximately 40% to approximately 70%, approximately 40% to approximately 60%, approximately 40% to approximately 50%, approximately 40% to approximately 45%, approximately 45% to approximately 800%, approximately 45% to approximately 750%, approximately 45% to approximately 700%, approximately 45% to approximately 650%, approximately 45% to approximately 600%, approximately 45% to approximately 550%, approximately 45% to approximately 500%, approximately 45% to approximately 450%, approximately 45% to about 400%, about 45% to about 350%, about 45% to about 300%, about 45% to about 280%, about 45% to about 260%, about 45% to about 240%, about 45% to about 220%, about 45% to about 200%, about 45% to about 180%, about 45% to about 160%, about 45% to about 140%, about 45% to about 120%, about 45% to about 100%, about 45% to about 90%, about 45% to about 80%, about 45% to about 70%, about 45% to about 60%, about 45% to about 50%, about 50% to about 800%, about 50% to about 750%, about 50% to about 700%, about 50% to about 650%, about 50% to about 600%, about 5 0% to about 550%, about 50% to about 500%, about 50% to about 450%, about 50% to about 400%, about 50% to about 350%, about 50% to about 300%, about 50% to about 280%, about 50% to about 260%, about 50% to about 240%, about 50% to about 220%, about 50% to about 200%, about 50% to about 180%, about 50% to about 160%, about 50% to about 140%, about 50% to about 120%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 800%, about 60% to about 750%, about 60% to about 700%, about 60% to about 650%, about 60% to about 600%, about 60% to about 550%, about 60% to about 500%, about 60% to about 450%, about 60% to about 400%, about 60% to about 350%, about 60% to about 300%, about 60% to about 280%, about 60% to about 260%, about 60% to about 240%, about 60% to about 220%, about 60% to about 200%, about 60% to about 180%, about 60% to about 160%, about 60% to about 140%, about 60% to about 120%, about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 800%, about 70% to about 750%.Approximately 70% to approximately 700%, approximately 70% to approximately 650%, approximately 70% to approximately 600%, approximately 70% to approximately 550%, approximately 70% to approximately 500%, approximately 70% to approximately 450%, approximately 70% to approximately 400%, approximately 70% to approximately 350%, approximately 70% to approximately 300%, approximately 70% to approximately 280%, approximately 70% to approximately 260%, approximately 70% to approximately 240%, approximately 70% to approximately 220%, approximately 70% to approximately 200%, approximately 70% to approximately 180%, approximately 70% to approximately 160%, approximately 70% to approximately 140%, approximately 70% to approximately 120%, approximately 70% to approximately 100%, approximately 70% to approximately 90%, approximately 70% to approximately 80%, approximately 80% to approximately 800%, approximately 80% to approximately 750%. Approximately 80% to approximately 700%, approximately 80% to approximately 650%, approximately 80% to approximately 600%, approximately 80% to approximately 550%, approximately 80% to approximately 500%, approximately 80% to approximately 450%, approximately 80% to approximately 400%, approximately 80% to approximately 350%, approximately 80% to approximately 300%, approximately 80% to approximately 280%, approximately 80% to approximately 260%, approximately 80% to approximately 240%, approximately 80% to approximately 220%, approximately 80% to approximately 200%, approximately 80% to approximately 180%, approximately 80% to approximately 160%, approximately 80% to approximately 140%, approximately 80% to approximately 120%, approximately 80% to approximately 100%, approximately 80% to approximately 90%, approximately 90% to approximately 800%, approximately 90% to approximately 750%, approximately 90% to approximately 70% 0%, about 90% to about 650%, about 90% to about 600%, about 90% to about 550%, about 90% to about 500%, about 90% to about 450%, about 90% to about 400%, about 90% to about 350%, about 90% to about 300%, about 90% to about 280%, about 90% to about 260%, about 90% to about 240%, about 90% to about 220%, about 90% to about 200%, about 90% to about 180%, about 90% to about 160%, about 90% to about 140%, about 90% to about 120%, about 90% to about 100%, about 100% to about 800%, about 100% to about 750%, about 100% to about 700%, about 100% to about 650%, about 100% to about 600%, about 100% to about 550%, about 100% to about 500%, about 100% to about 450%, about 100% to about 400%, about 100% to about 350%, about 100% to about 300%, about 100% to about 280%, about 100% to about 260%, about 100% to about 240%, about 100% to about 220%, about 100% to about 200%, about 100% to about 180%, about 100% to about 160%, about 100% to about 140%, about 100% to about 120%, about 120% to about 800%, about 120% to about 750%, about 120% to about 700%, about 120% to about 650%, about 120% to about 600%.About 120% to about 550%, about 120% to about 500%, about 120% to about 450%, about 120% to about 400%, about 120% to about 350%, about 120% to about 300%, about 120% to about 280%, about 120% to about 260%, about 120% to about 240%, about 120% to about 220%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 800%, about 140% to about 750%, about 140% to about 700%, about 140% to about 650%, about 140% to about 600%, about 140% to about 550%, about 140% to about 50% 0%, about 140% to about 450%, about 140% to about 400%, about 140% to about 350%, about 140% to about 300%, about 140% to about 280%, about 140% to about 260%, about 140% to about 240%, about 140% to about 220%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 800%, about 160% to about 750%, about 160% to about 700%, about 160% to about 650%, about 160% to about 600%, about 160% to about 550%, about 160% to about 500%, about 160% to about 450%, about 160% to about 400%, about 160% to Approximately 350%, approximately 160% to approximately 300%, approximately 160% to approximately 280%, approximately 160% to approximately 260%, approximately 160% to approximately 240%, approximately 160% to approximately 220%, approximately 160% to approximately 200%, approximately 160% to approximately 180%, approximately 180% to approximately 800%, approximately 180% to approximately 750%, approximately 180% to approximately 700%, approximately 180% to approximately 650%, approximately 180% to approximately 600%, approximately 180% to approximately 550%, approximately 180% to approximately 500%, approximately 180% to approximately 450%, approximately 180% to approximately 400%, approximately 180% to approximately 350%, approximately 180% to approximately 300%, approximately 180% to approximately 280%, approximately 180% to approximately 260%, approximately 18 0% to about 240%, about 180% to about 220%, about 180% to about 200%, about 200% to about 800%, about 200% to about 750%, about 200% to about 700%, about 200% to about 650%, about 200% to about 600%, about 200% to about 550%, about 200% to about 500%, about 200% to about 450%, about 200% to about 400%, about 200% to about 350%, about 200% to about 300%, about 200% to about 280%, about 200% to about 260%, about 200% to about 240%, about 200% to about 220%, about 220% to about 800%, about 220% to about 750%, about 220% to about 700%.Approximately 220% to approximately 650%, approximately 220% to approximately 600%, approximately 220% to approximately 550%, approximately 220% to approximately 500%, approximately 220% to approximately 450%, approximately 220% to approximately 400%, approximately 220% to approximately 350%, approximately 220% to approximately 300%, approximately 220% to approximately 280%, approximately 220% to approximately 260%, approximately 220% to approximately 240%, approximately 240% to approximately 800%, approximately 240% to approximately 750%, approximately 240% to approximately 700%, approximately 240% to approximately 650%, approximately 240% to approximately 600%, approximately 240% to approximately 550%, approximately 240% to approximately 500%, approximately 240% to approximately 450%, approximately 240% to approximately 400%, approximately 240% to approximately 350%. 0%, approximately 240% to approximately 300%, approximately 240% to approximately 280%, approximately 240% to approximately 260%, approximately 260% to approximately 800%, approximately 260% to approximately 750%, approximately 260% to approximately 700%, approximately 260% to approximately 650%, approximately 260% to approximately 600%, approximately 260% to approximately 550%, approximately 260% to approximately 500%, approximately 260% to approximately 450%, approximately 260% to approximately 400%, approximately 260% to approximately 350%, approximately 260% to approximately 300%, approximately 260% to approximately 280%, approximately 280% to approximately 800%, approximately 280% to approximately 750%, approximately 280% to approximately 700%, approximately 280% to approximately 650%, approximately 280% to approximately 600%, approximately 280% to Approximately 550%, approximately 280% to approximately 500%, approximately 280% to approximately 450%, approximately 280% to approximately 400%, approximately 280% to approximately 350%, approximately 280% to approximately 300%, approximately 300% to approximately 800%, approximately 300% to approximately 750%, approximately 300% to approximately 700%, approximately 300% to approximately 650%, approximately 300% to approximately 600%, approximately 300% to approximately 550%, approximately 300% to approximately 500%, approximately 300% to approximately 450%, approximately 300% to approximately 400%, approximately 300% to approximately 350%, approximately 350% to approximately 800%, approximately 350% to approximately 750%, approximately 350% to approximately 700%, approximately 350% to approximately 650%, approximately 350% to approximately 600%, approximately 35 0% to about 550%, about 350% to about 500%, about 350% to about 450%, about 350% to about 400%, about 400% to about 800%, about 400% to about 750%, about 400% to about 700%, about 400% to about 650%, about 400% to about 600%, about 400% to about 550%, about 400% to about 500%, about 400% to about 450%, about 450% to about 800%, about 450% to about 750%, about 450% to about 700%, about 450% to about 650%, about 450% to about 600%, about 450% to about 550%, about 450% to about 500%, about 500% to about 800%, about 500% to about 750%.Approximately 500% to 700%, approximately 500% to 650%, approximately 500% to 600%, approximately 500% to 550%, approximately 550% to 800%, approximately 550% to 750%, approximately 550% to 700%, approximately 550% to 650%, approximately 550% to 600%, approximately 600% to 800%, approximately 600% to 750%, approximately 600% to 700%, approximately 600% to 650%, approximately 650% to 800%, approximately 650% to 750%, approximately 650% to 700%, approximately 700% to 800%, approximately 700% to 750%, or approximately 750% to 800% (e.g., compared to the levels of one or more cytokines, chemokines, cytotoxic particles, and regulatory molecules in a control that has not been exposed to any of the multi-chain chimeric peptides described herein).

[0256] Methods to induce or increase the proliferation of immune cells

[0257] This document also provides methods for inducing or increasing the proliferation of immune cells (e.g., any exemplary immune cells described herein or known in the art), methods comprising contacting the immune cells with an effective amount of any multi-chain chimeric polypeptide described herein or any composition described herein (e.g., a pharmaceutical composition). In some instances, the immune cells are contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to induce immune cell stimulation).

[0258] In some instances, the immune cells have been previously obtained from the subject (e.g., a mammal, such as a human). Some embodiments of these methods further include obtaining the immune cells from the subject prior to the contact step.

[0259] In some instances, immune cells are brought into contact in vivo. In such embodiments, the multi-chain chimeric polypeptide is administered to the subject in an amount sufficient to stimulate immune cells in the subject (e.g., a mammal, such as a human).

[0260] In some instances of any of the methods described herein, immune cells may be immature thymocytes, peripheral blood lymphocytes, primary T cells, pluripotent Th cell precursors, lymphoid progenitor cells, Treg cells, memory T cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8+ cells, etc. + T cells, CD4 + T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, or natural killer cells, or combinations thereof. In some embodiments, the T cells may be progenitor-depleted CD8 progenitor cells. + (Tpex) cells (e.g., CD3)+ CD8 + TCF1 + TOX + PD1 + TIGIT + CD27 + Cellular and effector memory CD8 + T (Tem) cells (e.g., CD3) + CD8 + CD44 (high CD62L, low cellularity), central memory CD8 + T (Tcm) cells (e.g., CD3) + CD8 + CD44 (high CD62L high cellularity), virtual memory CD8 + T (Tvm) cells (e.g., CD3) + CD8 + CD44 high CD49d low cellularity), antigen-experienced CD8 + T cells (e.g., CD44) + CCR7 + CD127 + CD122 + CD27 + CD62L + perforin + CD103 + CD69 + Cellular cells, terminally exhausted CD8 + T (Tex) cells (e.g., TOX) + TCF1 - Granulase B + PD-1 + Tim-3 + CD101 + CXCR3 + CCR5 + (cells) or memory stem CD8 + T (Tscm) cells (e.g., CD3) + CD8 + CD45RA, CCR7 + CD27 + CD95 + CXCR3 + cell).

[0261] In some instances, the immune cells have been previously genetically modified to express chimeric antigen receptors or recombinant T-cell receptors. In some instances, the immune cells (e.g., any immune cells described herein) have been previously genetically modified to express co-stimulatory molecules (e.g., CD28).

[0262] Some embodiments of these methods may further include, after the contact step, introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T-cell receptor into an immune cell (e.g., any immune cell described herein). Some embodiments of these methods may further include, after the contact step, introducing a nucleic acid encoding a co-stimulatory molecule (e.g., CD28) into an immune cell (e.g., any immune cell described herein).

[0263] Some embodiments of these methods may further include administering a therapeutically effective amount of immune cells to a subject in need (e.g., any of the exemplary subjects described herein).

[0264] In some instances, participants may be those who have been identified or diagnosed with an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

[0265] In some instances, participants may be those who have been identified or diagnosed with cancer. Non-limiting examples of cancer include: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal 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.

[0266] In some instances, the subject may be a subject who has been diagnosed or identified with an infectious disease. Non-limiting examples of infectious diseases include infections with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus and hepatitis C virus, human papillomavirus, or influenza virus.

[0267] The detection of immune cell proliferation can be performed using methods known in the art, such as cell counting (e.g., fluorescence-assisted flow cytometry), microscopy, and immunofluorescence microscopy, for example, by comparing the rate of increase in immune cell concentration in a sample untouched by the multi-chain chimeric peptide with the rate of increase in immune cell concentration in a similar sample contacted with any of the multi-chain chimeric peptides described herein.

[0268] In other instances, immune cell proliferation can be indirectly detected by detecting an increase in the levels of one or more cytokines, chemokines, cytotoxic particles, or regulatory molecules (e.g., one or more of IL-2, IFN-γ, IL-1, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-33, leukotriene B4, CCL5, TNFα, granzyme, perforin, TGFβ, STAT3, STAT4, STAT5, RORKT, FOXP3, and GATA3) secreted or upregulated by proliferating immune cells (e.g., compared to the levels of one or more cytokines, chemokines, cytotoxic particles, and regulatory molecules in a control that has not been exposed to any of the multi-chain chimeric peptides described herein).

[0269] In some embodiments, the methods provided herein may result in an increased rate of increase in immune cell concentration in samples contacted with any of the multi-chain chimeric peptides described herein (e.g., an increase of about 1% to about 800%, or any subrange of that range described herein), compared to the rate of increase in immune cell concentration in similar control samples that have not been contacted with any of the multi-chain chimeric peptides described herein.

[0270] Methods to induce immune cell differentiation

[0271] This document also provides a method for inducing immune cells (e.g., any exemplary immune cells described herein or known in the art) to differentiate into memory or memory-like immune cells, the method comprising contacting the immune cells with an effective amount of any multi-chain chimeric polypeptide described herein or any composition described herein (e.g., a pharmaceutical composition). In some instances, the immune cells are contacted in vitro (e.g., in a suitable liquid culture medium under conditions sufficient to induce immune cell stimulation).

[0272] In some instances, the immune cells have been previously obtained from the subject (e.g., a mammal, such as a human). Some embodiments of these methods further include obtaining the immune cells from the subject prior to the contact step.

[0273] In some instances, immune cells are brought into contact in vivo. In such embodiments, the multi-chain chimeric polypeptide is administered to the subject in an amount sufficient to stimulate immune cells in the subject (e.g., a mammal, such as a human).

[0274] In some instances of any of the methods described herein, the immune cells may be immature thymocytes, peripheral blood lymphocytes, primary T cells, pluripotent Th cell precursors, lymphoid progenitor cells, Treg cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8+ T cells, CD4+ T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, or natural killer cells or combinations thereof.

[0275] In some instances, the immune cells have been previously genetically modified to express chimeric antigen receptors or recombinant T-cell receptors. In some instances, the immune cells (e.g., any immune cells described herein) have been previously genetically modified to express co-stimulatory molecules (e.g., CD28).

[0276] In some instances, an effective amount of any multi-chain chimeric polypeptide or any composition described herein (e.g., a pharmaceutical composition) is combined with an anti-TF IgG1 antibody to generate memory or memory-like immune cells.

[0277] Some embodiments of these methods may further include, after the contact step, introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T-cell receptor into an immune cell (e.g., any immune cell described herein). Some embodiments of these methods may further include, after the contact step, introducing a nucleic acid encoding a co-stimulatory molecule (e.g., CD28) into an immune cell (e.g., any immune cell described herein).

[0278] Some embodiments of these methods may further include administering a therapeutically effective amount of immune cells to a subject in need (e.g., any of the exemplary subjects described herein).

[0279] In some instances, participants may be those who have been identified or diagnosed with an age-related disease or condition. Non-limiting examples of age-related diseases or disorders include: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

[0280] In some instances, participants may be those who have been identified or diagnosed with cancer. Non-limiting examples of cancer include: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal 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.

[0281] In some instances, the subject may be a subject who has been diagnosed or identified with an infectious disease. Non-limiting examples of infectious diseases include infections with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus and hepatitis C virus, human papillomavirus, or influenza virus.

[0282] In some instances, the immune cells are NK cells, and the detection of memory NK cells may include, for example, detecting the levels of one or more of the following: IL-12, IL-18, IL-33, CD25, CD69, CD62L, STAT4, Zbtb32, DNAM-1, NKp30, NKp44, NKp46, BIM, Noxa, SOCS1, BNIP3, BNIP3L, IFN-γ, CXCL16, CXCR6, NKG2D, TRAIL, CD49, Ly49D, CD49b, and Ly79H. The following literature describes NK memory cells and their detection methods: O'Sullivan et al., Immunity 43:634-645, 2015.

[0283] In some instances, the immune cells are T cells, and the detection of memory T cells may include, for example, detecting the expression levels of one or more of CD45RO, CCR7, L-selectin (CD62L), CD44, CD45RA, integrin αeβ7, CD43, CD27, CD28, IL-7Rα, CD95, IL-2Rβ, CXCR3, and LFA-1. In some instances, the immune cells are B cells, and the detection of memory B cells may include, for example, detecting the expression level of CD27. Other types and markers of memory or memory-like immune cells are known in the art.

[0284] Treatment

[0285] This document also provides methods for treating a subject in need (e.g., any exemplary subject described herein or known in the art), including administering to the subject a therapeutically effective amount of any multi-chain chimeric polypeptide described herein or any composition described herein (e.g., a pharmaceutical composition).

[0286] In some embodiments of these methods, the subject has been identified or diagnosed with cancer. Non-limiting examples of cancer include: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal 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, these methods may result in a reduction in the number, severity, or frequency of one or more symptoms of cancer in a subject (e.g., compared to the number, severity, or frequency of one or more symptoms of cancer in a subject before treatment). In some embodiments, these methods can result in a reduction in the volume of one or more solid tumors in a subject (e.g., a reduction of about 1% to about 99%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 99%). Small, approximately 5% decrease to approximately 95% decrease, approximately 5% decrease to approximately 90% decrease, approximately 5% decrease to approximately 85% decrease, approximately 5% decrease to approximately 80% decrease, approximately 5% decrease to approximately 75% decrease, approximately 5% decrease to approximately 70% decrease, approximately 5% decrease to approximately 65% ​​decrease, approximately 5% decrease to approximately 60% decrease, approximately 5% decrease to approximately 55% decrease, approximately 5% decrease to approximately 50% decrease, approximately 5% decrease to approximately 45% decrease, approximately 5% decrease to approximately 40% decrease, approximately 5% decrease to approximately 35% decrease, approximately 5% decrease to approximately 30% decrease, approximately 5% decrease to approximately 25% decrease, approximately 5% decrease to approximately 20% decrease, approximately 5% decrease to approximately 15% decrease, approximately 5% decrease to approximately 10% decrease, approximately 10% decrease to approximately 99% decrease, approximately 10% decrease to approximately 95% decrease, approximately 10% decrease to approximately 90% decrease, approximately 10% decrease to approximately 85% decrease, approximately 10% decrease to approximately 80% decrease.Approximately 10% decrease to approximately 75% decrease, approximately 10% decrease to approximately 70% decrease, approximately 10% decrease to approximately 65% ​​decrease, approximately 10% decrease to approximately 60% decrease, approximately 10% decrease to approximately 55% decrease, approximately 10% decrease to approximately 50% decrease, approximately 10% decrease to approximately 45% decrease, approximately 10% decrease to approximately 40% decrease, approximately 10% decrease to approximately 35% decrease, approximately 10% decrease to approximately 30% decrease, approximately 10% decrease to approximately 25% decrease, approximately 10% decrease to approximately 20% decrease, approximately 10% decrease to approximately 15% decrease, approximately 15% decrease to approximately 99% decrease, approximately 15% decrease to approximately 95% decrease, approximately 15% decrease to approximately 90% decrease, approximately 15% decrease to approximately 85% decrease, approximately 15% decrease to approximately 80% decrease. % decrease, approximately 15% decrease to approximately 75% decrease, approximately 15% decrease to approximately 70% decrease, approximately 15% decrease to approximately 65% ​​decrease, approximately 15% decrease to approximately 60% decrease, approximately 15% decrease to approximately 55% decrease, approximately 15% decrease to approximately 50% decrease, approximately 15% decrease to approximately 45% decrease, approximately 15% decrease to approximately 40% decrease, approximately 15% decrease to approximately 35% decrease, approximately 15% decrease to approximately 30% decrease, approximately 15% decrease to approximately 25% decrease, approximately 15% decrease to approximately 20% decrease, approximately 20% decrease to approximately 99% decrease, approximately 20% decrease to approximately 95% decrease, approximately 20% decrease to approximately 90% decrease, approximately 20% decrease to approximately 85% decrease, approximately 20% decrease to approximately 80% decrease, approximately 20% decrease to Approximately 75% reduction, approximately 20% reduction to approximately 70% reduction, approximately 20% reduction to approximately 65% ​​reduction, approximately 20% reduction to approximately 60% reduction, approximately 20% reduction to approximately 55% reduction, approximately 20% reduction to approximately 50% reduction, approximately 20% reduction to approximately 45% reduction, approximately 20% reduction to approximately 40% reduction, approximately 20% reduction to approximately 35% reduction, approximately 20% reduction to approximately 30% reduction, approximately 20% reduction to approximately 25% reduction, approximately 25% reduction to approximately 99% reduction, approximately 25% reduction to approximately 95% reduction, approximately 25% reduction to approximately 90% reduction, approximately 25% reduction to approximately 85% reduction, approximately 25% reduction to approximately 80% reduction, approximately 25% reduction to approximately 75% reduction, approximately 25% reduction to approximately 70% reduction, approximately 25 % decrease to about 65% decrease, about 25% decrease to about 60% decrease, about 25% decrease to about 55% decrease, about 25% decrease to about 50% decrease, about 25% decrease to about 45% decrease, about 25% decrease to about 40% decrease, about 25% decrease to about 35% decrease, about 25% decrease to about 30% decrease, about 30% decrease to about 99% decrease, about 30% decrease to about 95% decrease, about 30% decrease to about 90% decrease, about 30% decrease to about 85% decrease, about 30% decrease to about 80% decrease, about 30% decrease to about 75% decrease, about 30% decrease to about 70% decrease, about 30% decrease to about 65% decrease, about 30% decrease to about 60% decrease, about 30% decrease to about 55% decrease.Approximately 30% decreases to approximately 50% decreases, approximately 30% decreases to approximately 45% decreases, approximately 30% decreases to approximately 40% decreases, approximately 30% decreases to approximately 35% decreases, approximately 35% decreases to approximately 99% decreases, approximately 35% decreases to approximately 95% decreases, approximately 35% decreases to approximately 90% decreases, approximately 35% decreases to approximately 85% decreases, approximately 35% decreases to approximately 80% decreases, approximately 35% decreases to approximately 75% decreases, approximately 35% decreases to approximately 70% decreases, approximately 35% decreases to approximately 65% ​​decreases, approximately 35% decreases to approximately 60% decreases, approximately 35% decreases to approximately 55% decreases, approximately 35% decreases to approximately 50% decreases, approximately 35% decreases to approximately 45% decreases, approximately 35% decreases to approximately 40% decreases, approximately 40% decreases to approximately 99% decreases. % decrease, approximately 40% decrease to approximately 95% decrease, approximately 40% decrease to approximately 90% decrease, approximately 40% decrease to approximately 85% decrease, approximately 40% decrease to approximately 80% decrease, approximately 40% decrease to approximately 75% decrease, approximately 40% decrease to approximately 70% decrease, approximately 40% decrease to approximately 65% ​​decrease, approximately 40% decrease to approximately 60% decrease, approximately 40% decrease to approximately 55% decrease, approximately 40% decrease to approximately 50% decrease, approximately 40% decrease to approximately 45% decrease, approximately 45% decrease to approximately 99% decrease, approximately 45% decrease to approximately 95% decrease, approximately 45% decrease to approximately 90% decrease, approximately 45% decrease to approximately 85% decrease, approximately 45% decrease to approximately 80% decrease, approximately 45% decrease to approximately 75% decrease, approximately 45% decrease to Approximately 70% reduction, approximately 45% reduction to approximately 65% ​​reduction, approximately 45% reduction to approximately 60% reduction, approximately 45% reduction to approximately 55% reduction, approximately 45% reduction to approximately 50% reduction, approximately 50% reduction to approximately 99% reduction, approximately 50% reduction to approximately 95% reduction, approximately 50% reduction to approximately 90% reduction, approximately 50% reduction to approximately 85% reduction, approximately 50% reduction to approximately 80% reduction, approximately 50% reduction to approximately 75% reduction, approximately 50% reduction to approximately 70% reduction, approximately 50% reduction to approximately 65% ​​reduction, approximately 50% reduction to approximately 60% reduction, approximately 50% reduction to approximately 55% reduction, approximately 55% reduction to approximately 99% reduction, approximately 55% reduction to approximately 95% reduction, approximately 55% reduction to approximately 90% reduction, approximately 55 % decrease to about 85% decrease, about 55% decrease to about 80% decrease, about 55% decrease to about 75% decrease, about 55% decrease to about 70% decrease, about 55% decrease to about 65% decrease, about 55% decrease to about 60% decrease, about 60% decrease to about 99% decrease, about 60% decrease to about 95% decrease, about 60% decrease to about 90% decrease, about 60% decrease to about 85% decrease, about 60% decrease to about 80% decrease, about 60% decrease to about 75% decrease, about 60% decrease to about 70% decrease, about 60% decrease to about 65% decrease, about 65% decrease to about 99% decrease, about 65% decrease to about 95% decrease, about 65% decrease to about 90% decrease, about 65% decrease to about 85% decrease.Approximately 65% ​​decreased to approximately 80% decrease, approximately 65% ​​decreased to approximately 75% decrease, approximately 65% ​​decreased to approximately 70% decrease, approximately 70% decreased to approximately 99% decrease, approximately 70% decreased to approximately 95% decrease, approximately 70% decreased to approximately 90% decrease, approximately 70% decreased to approximately 85% decrease, approximately 70% decreased to approximately 80% decrease, approximately 70% decreased to approximately 75% decrease, approximately 75% decreased to approximately 99% decrease, approximately 75% decreased to approximately 95% decrease, approximately 75% decreased to approximately 90% decrease, approximately 75% decreased to approximately 85% decrease. The reduction is approximately 75% to approximately 80%, approximately 80% to approximately 99%, approximately 80% to approximately 95%, approximately 80% to approximately 90%, approximately 80% to approximately 85%, approximately 85% to approximately 99%, approximately 85% to approximately 95%, approximately 85% to approximately 90%, approximately 90% to approximately 99%, approximately 90% to approximately 95%, or approximately 95% to approximately 99% (e.g., compared to the volume of one or more solid tumors before or at the start of treatment). In some embodiments, these methods may reduce (e.g., by approximately 1% to approximately 99%, or any subrange of this range described herein) the risk of metastasis or one or more additional metastases in a subject (e.g., compared to the risk of metastasis or one or more additional metastases in a subject before treatment or in a similar subject population or a group of subjects who received different treatments).

[0287] In some instances of these methods, the subjects have been identified or diagnosed with age-related diseases or conditions. Non-limiting examples of age-related diseases and conditions include Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some instances, these methods can lead to a reduction in the number, severity, or frequency of one or more symptoms of age-related diseases or conditions in a subject (e.g., compared to the number, severity, or frequency of one or more symptoms of age-related diseases or conditions in the subject before treatment). In some instances, for example, these methods can result in a reduction in the number of senescent cells in a subject compared to the number of senescent cells in the subject before treatment (e.g., a reduction in the number of senescent cells in one or more specific tissues involved in and / or associated with age-related diseases or disorders) (e.g., a reduction of about 1% to about 99%, about 1% to about 95%, about 1% to about 90%, about 1% to about 85%, about 1% to about 80%, about 1% to about 75%, about 1% to about 70%, about 1% to about 65%, about 1% to about 60%, about 1% to about 55%, about 1% to about 50%, about 1% to about 45%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 30%). The percentages of the following reductions are listed: approximately 25%, approximately 1%, approximately 1%, approximately 15%, approximately 1%, approximately 10%, approximately 1%, approximately 5%, approximately 5%, approximately 99%, approximately 5%, approximately 95%, approximately 5%, approximately 90%, approximately 85%, approximately 80%, approximately 75%, approximately 70%, approximately 65%, approximately 60%, approximately 5%, approximately 55%, approximately 50%, approximately 45%, approximately 40%, approximately 35%, approximately 30%, approximately 25%, approximately 20%, approximately 15%, and approximately 10%.Approximately 10% reduction to approximately 99% reduction, approximately 10% reduction to approximately 95% reduction, approximately 10% reduction to approximately 90% reduction, approximately 10% reduction to approximately 85% reduction, approximately 10% reduction to approximately 80% reduction, approximately 10% reduction to approximately 75% reduction, approximately 10% reduction to approximately 70% reduction, approximately 10% reduction to approximately 65% ​​reduction, approximately 10% reduction to approximately 60% reduction, approximately 10% reduction to approximately 55% reduction, approximately 10% reduction to approximately 50% reduction, approximately 10% reduction to approximately 45% reduction, approximately 10% reduction to approximately 40% reduction, approximately 10% reduction to approximately 35% reduction, approximately 10% reduction to approximately 30% reduction, approximately 10% reduction to approximately 25% reduction, approximately 10% reduction to approximately 20% reduction, approximately 10% reduction to approximately 15% reduction. Approximately 15% reduced to approximately 99% reduction, approximately 15% reduced to approximately 95% reduction, approximately 15% reduced to approximately 90% reduction, approximately 15% reduced to approximately 85% reduction, approximately 15% reduced to approximately 80% reduction, approximately 15% reduced to approximately 75% reduction, approximately 15% reduced to approximately 70% reduction, approximately 15% reduced to approximately 65% ​​reduction, approximately 15% reduced to approximately 60% reduction, approximately 15% reduced to approximately 55% reduction, approximately 15% reduced to approximately 50% reduction, approximately 15% reduced to approximately 45% reduction, approximately 15% reduced to approximately 40% reduction, approximately 15% reduced to approximately 35% reduction, approximately 15% reduced to approximately 30% reduction, approximately 15% reduced to approximately 25% reduction, approximately 15% reduced to approximately 20% reduction, approximately 20% reduced to approximately 99% reduction. Approximately 20% reduced to approximately 95% reduction, approximately 20% reduced to approximately 90% reduction, approximately 20% reduced to approximately 85% reduction, approximately 20% reduced to approximately 80% reduction, approximately 20% reduced to approximately 75% reduction, approximately 20% reduced to approximately 70% reduction, approximately 20% reduced to approximately 65% ​​reduction, approximately 20% reduced to approximately 60% reduction, approximately 20% reduced to approximately 55% reduction, approximately 20% reduced to approximately 50% reduction, approximately 20% reduced to approximately 45% reduction, approximately 20% reduced to approximately 40% reduction, approximately 20% reduced to approximately 35% reduction, approximately 20% reduced to approximately 30% reduction, approximately 20% reduced to approximately 25% reduction, approximately 25% reduced to approximately 99% reduction, approximately 25% reduced to approximately 95% reduction, approximately 25% reduced to approximately 90% reduction. Approximately 25% reduced to approximately 85% reduction, approximately 25% reduced to approximately 80% reduction, approximately 25% reduced to approximately 75% reduction, approximately 25% reduced to approximately 70% reduction, approximately 25% reduced to approximately 65% ​​reduction, approximately 25% reduced to approximately 60% reduction, approximately 25% reduced to approximately 55% reduction, approximately 25% reduced to approximately 50% reduction, approximately 25% reduced to approximately 45% reduction, approximately 25% reduced to approximately 40% reduction, approximately 25% reduced to approximately 35% reduction, approximately 25% reduced to approximately 30% reduction, approximately 30% reduced to approximately 99% reduction, approximately 30% reduced to approximately 95% reduction, approximately 30% reduced to approximately 90% reduction, approximately 30% reduced to approximately 85% reduction, approximately 30% reduced to approximately 80% reduction, approximately 30% reduced to approximately 75% reduction.Approximately 30% to approximately 70% reduction, approximately 30% to approximately 65% ​​reduction, approximately 30% to approximately 60% reduction, approximately 30% to approximately 55% reduction, approximately 30% to approximately 50% reduction, approximately 30% to approximately 45% reduction, approximately 30% to approximately 40% reduction, approximately 30% to approximately 35% reduction, approximately 35% to approximately 99% reduction, approximately 35% to approximately 95% reduction, approximately 35% to approximately 90% reduction, approximately 35% to approximately 85% reduction, approximately 35% to approximately 80% reduction, approximately 35% to approximately 75% reduction, approximately 35% to approximately 70% reduction, approximately 35% to approximately 65% ​​reduction, approximately 35% to approximately 60% reduction, approximately 35% to approximately 55% reduction. Approximately 35% decreased to approximately 50% decrease, approximately 35% decreased to approximately 45% decrease, approximately 35% decreased to approximately 40% decrease, approximately 40% decreased to approximately 99% decrease, approximately 40% decreased to approximately 95% decrease, approximately 40% decreased to approximately 90% decrease, approximately 40% decreased to approximately 85% decrease, approximately 40% decreased to approximately 80% decrease, approximately 40% decreased to approximately 75% decrease, approximately 40% decreased to approximately 70% decrease, approximately 40% decreased to approximately 65% ​​decrease, approximately 40% decreased to approximately 60% decrease, approximately 40% decreased to approximately 55% decrease, approximately 40% decreased to approximately 45% decrease, approximately 45% decreased to approximately 99% decrease, approximately 45% decreased to approximately 95% decrease, approximately 45% decreased to approximately 90% decrease Approximately 45% to approximately 85% reduction, approximately 45% to approximately 80% reduction, approximately 45% to approximately 75% reduction, approximately 45% to approximately 70% reduction, approximately 45% to approximately 65% ​​reduction, approximately 45% to approximately 60% reduction, approximately 45% to approximately 55% reduction, approximately 45% to approximately 50% reduction, approximately 50% to approximately 99% reduction, approximately 50% to approximately 95% reduction, approximately 50% to approximately 90% reduction, approximately 50% to approximately 85% reduction, approximately 50% to approximately 80% reduction, approximately 50% to approximately 75% reduction, approximately 50% to approximately 70% reduction, approximately 50% to approximately 65% ​​reduction, approximately 50% to approximately 60% reduction, approximately 50% to approximately 55% reduction Approximately 55% reduced to approximately 99% reduction, approximately 55% reduced to approximately 95% reduction, approximately 55% reduced to approximately 90% reduction, approximately 55% reduced to approximately 85% reduction, approximately 55% reduced to approximately 80% reduction, approximately 55% reduced to approximately 75% reduction, approximately 55% reduced to approximately 70% reduction, approximately 55% reduced to approximately 65% ​​reduction, approximately 55% reduced to approximately 60% reduction, approximately 60% reduced to approximately 99% reduction, approximately 60% reduced to approximately 95% reduction, approximately 60% reduced to approximately 90% reduction, approximately 60% reduced to approximately 85% reduction, approximately 60% reduced to approximately 80% reduction, approximately 60% reduced to approximately 75% reduction, approximately 60% reduced to approximately 70% reduction, approximately 60% reduced to approximately 65% ​​reduction, approximately 65% ​​reduced to approximately 99% reduction.Approximately 65% ​​decreased to approximately 95% decrease, approximately 65% ​​decreased to approximately 90% decrease, approximately 65% ​​decreased to approximately 85% decrease, approximately 65% ​​decreased to approximately 80% decrease, approximately 65% ​​decreased to approximately 75% decrease, approximately 65% ​​decreased to approximately 70% decrease, approximately 70% decreased to approximately 99% decrease, approximately 70% decreased to approximately 95% decrease, approximately 70% decreased to approximately 90% decrease, approximately 70% decreased to approximately 85% decrease, approximately 70% decreased to approximately 80% decrease, approximately 70% decreased to approximately 75% decrease, approximately 75% decreased to approximately 99% decrease, approximately 75% decreased to approximately 95% reduction, approximately 75% reduction to approximately 90% reduction, approximately 75% reduction to approximately 85% reduction, approximately 75% reduction to approximately 80% reduction, approximately 80% reduction to approximately 99% reduction, approximately 80% reduction to approximately 95% reduction, approximately 80% reduction to approximately 90% reduction, approximately 80% reduction to approximately 85% reduction, approximately 85% reduction to approximately 99% reduction, approximately 85% reduction to approximately 90% reduction, approximately 90% reduction to approximately 99% reduction, approximately 90% reduction to approximately 95% reduction or approximately 95% reduction to approximately 99% reduction.

[0288] In some instances of these methods, the subject has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious diseases include infections with human immunodeficiency virus (HIV), cytomegalovirus (CMV), adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, human papillomavirus (HPV), and influenza virus. In some embodiments, these methods may result in a reduction in the infection titer (e.g., viral titer) in the subject (e.g., compared to the infection titer in the subject before treatment). In some embodiments, these methods may result in a reduction in the number, severity, or frequency of one or more symptoms of an infectious disease (e.g., viral infection) in the subject (e.g., compared to the number, severity, or frequency of one or more symptoms of an infectious disease in the subject before treatment).

[0289] The term "subject" refers to any mammal. In some embodiments, the subject or "subject requiring treatment" may be a canine (e.g., a dog), a feline (e.g., a cat), an equine (e.g., a horse), a sheep, a cow, a pig, a goat, a primate such as an ape (e.g., a monkey (e.g., a marmoset, a baboon) or an ape (e.g., a gorilla, a chimpanzee, an orangutan, or a gibbon) or a human; or a rodent (e.g., a mouse, a guinea pig, a hamster, or a rat). In some embodiments, the subject or "subject requiring treatment" may be a non-human mammal, particularly a mammal commonly used as a model to demonstrate therapeutic efficacy in humans (e.g., a mouse, a rabbit, a pig, a dog, or a primate).

[0290] Methods to kill cancer cells, infected cells, or senescent cells

[0291] This document also provides methods for killing cancer cells (e.g., any exemplary cancer type described herein or known in the art), infected cells (e.g., cells infected by any of the exemplary viruses described herein or known in the art), or senescent cells (e.g., senescent cancer cells, senescent fibroblasts, or senescent endothelial cells) in a subject in need (e.g., any exemplary subject described herein or known in the art), methods comprising administering to the subject a therapeutically effective amount of any multi-chain chimeric polypeptide described herein or any composition described herein (e.g., a pharmaceutical composition).

[0292] In some embodiments of these methods, the subject has been identified or diagnosed with cancer. Non-limiting examples of cancer include: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal 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.

[0293] In some instances of these methods, the subjects have been identified or diagnosed with age-related diseases or conditions. Non-limiting examples of age-related diseases and conditions include Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.

[0294] In some instances of these methods, the subject has been diagnosed or identified as having an infectious disease. Non-limiting examples of infectious diseases include infections with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus and hepatitis C virus, human papillomavirus, and influenza virus.

[0295] Senescent cells

[0296] Senescence is a form of irreversible growth arrest accompanied by phenotypic changes, resistance to apoptosis, and activation of damage-sensing signaling pathways. Cellular senescence was initially described in cultured human fibroblasts, which lose their proliferative capacity and reach permanent arrest after approximately 50 population doublings (known as the Hayflick limit). Senescence is considered to be a stress response induced by a wide range of intrinsic and extrinsic damage, including oxidative and genotoxic stress, DNA damage, telomere loss, oncogenic activation, mitochondrial dysfunction, or chemotherapeutic agents.

[0297] Senescent cells retain metabolic activity and can influence tissue hemostasis, disease, and aging through their secretory phenotype. Senescence is considered a physiological process and is important for promoting wound healing, tissue homeostasis, regeneration, and regulating fibrosis. For example, transient induction of senescent cells has been observed during wound healing, and they contribute to wound healing. One of the most important roles of senescence may be its role in tumor suppression. However, the accumulation of senescent cells also drives aging and age-related diseases and conditions. The senescence phenotype can also trigger chronic inflammatory responses and thus enhance chronic inflammatory conditions to promote tumor growth. The link between senescence and aging was initially based on the observation of the accumulation of senescent cells in aging tissues. The use of transgenic models has enabled the systematic detection of senescent cells in many age-related pathologies. Strategies for selectively eliminating senescent cells have demonstrated that senescent cells can indeed play a causal role in aging and related pathologies.

[0298] Senescent cells exhibit important and unique characteristics, including changes in morphology, chromatin organization, gene expression, and metabolism. Several biochemical and functional properties associated with cellular senescence are present, such as (i) increased expression of cyclin-dependent kinase repressors p16 and p21, (ii) the presence of senescence-associated β-galactosidase, a marker of lysosomal activity, (iii) the appearance of senescence-associated heterochromatin foci and downregulation of laminin B1 levels, (iv) resistance to apoptosis induced by increased expression of anti-apoptotic BCL family proteins, and (v) upregulation of CD26 (DPP4), CD36 (scavenger receptor), forkhead box 4 (FOXO4), and secretory carrier membrane protein 4 (SCAMP4). Senescent cells also express inflammatory features, known as the senescence-associated secretory phenotype (SASP). Through senescent cytokines (SASPs), senescent cells produce a wide range of inflammatory cytokines (IL-6, IL-8), growth factors (TGF-β), chemokines (CCL-2), and matrix metalloproteinases (MMP-3, MMP-9), which operate autonomously to enhance senescence (autocrine function) and communicate with and alter the microenvironment (paracrine function). SASP factors can promote tumor suppression by triggering senescence surveillance, i.e., immune-mediated clearance of senescent cells. However, chronic inflammation is also a known driver of tumorigenesis, and mounting evidence suggests that chronic SASPs may also promote cancer and age-related diseases.

[0299] The secretory profile of senescent cells is environmentally dependent. For example, mitochondrial dysfunction-associated senescence (MiDAS) induced by different mitochondrial dysfunctions in human fibroblasts leads to the emergence of senescent inflammatory cytokines (SASPs) lacking IL-1-dependent inflammatory factors. A decreased NAD+ / NADH ratio activates AMPK signaling, which induces MiDAS by activating p53. Thus, p53 inhibits NF-κB signaling, a key inducing factor for pro-inflammatory SASPs. Conversely, cellular senescence induced by persistent DNA damage in human cells induces inflammatory SASPs dependent on the activation of ataxia-telangiectasia mutant (ATM) kinase, but not on p53 activation. In particular, the expression and secretion levels of IL-6 and IL-8 are increased. Cellular senescence induced by ectopic expression of p16INK4a and p21CIP1 has also been demonstrated to induce a senescent phenotype in human fibroblasts in the absence of inflammatory SASPs, suggesting that growth arrest itself does not stimulate SASPs.

[0300] One of the most prominent characteristics of senescence is stable growth arrest. This is achieved through two important pathways, namely p16 / Rb and p53 / p21, both of which are key to tumor suppression. DNA damage leads to: (1) high deposition of γH2Ax (a histone-encoding gene) and 53BP1 (involved in the DNA damage response) in chromatin: this leads to activation of the kinase cascade, which ultimately results in p53 activation; and (2) activation of p16INK4a and ARF (both encoded by CDKN2A) and p15INK4b (encoded by CDKN2B): p53 induces transcription of cyclin-dependent kinase inhibitors (p21) and, together with p16INK4a and p15INK4b, blocks genes (CDK4 and CDK6) used for cell cycle progression. This ultimately leads to insufficient phosphorylation of retinoblastoma protein (Rb) and arrests the cell cycle in the G1 phase.

[0301] In normal aging, selective killing of senescent cells has been shown to significantly increase healthy lifespan in mice and improve outcomes of age-related diseases or cancer therapies (Ovadya, J Clin Invest. [Journal of Clinical Research] 128(4):1247-1254, 2018). In nature, senescent cells are typically eliminated by innate immune cells. Induction of senescence not only prevents the potential proliferation and transformation of damaged / altered cells but also promotes tissue repair by producing SASP factors (which primarily act as chemical inducers for natural killer (NK) cells, such as IL-15 and CCL2, and for macrophages, such as CFS-1 and CCL2). These innate immune cells mediate immune surveillance mechanisms that eliminate stress cells. Senescent cells typically upregulate NK cell activation receptors NKG2D and DNAM-1 ligands, which belong to the stress-inducible ligand family: important components of first-line immunity in the defense against infectious diseases and malignancies. Upon receptor activation, NK cells can then specifically induce the death of senescent cells through their cytolytic mechanisms. The role of NK cells in the immune surveillance of senescent cells has been demonstrated in liver fibrosis (Sagiv, Oncogene 32(15): 1971-1977, 2013), hepatocellular carcinoma (Iannello, J Exp Med 210(10): 2057-2069, 2013), multiple myeloma (Soriani, Blood 113(15): 3503-3511, 2009), and glioma cells stressed by mevalonate pathway dysfunction (Ciaglia, Int J Cancer 142(1): 176-190, 2018). Endometrial cells undergo acute cellular senescence and do not differentiate into decidual cells. Differentiated decidual cells secrete IL-15, thereby recruiting uterine NK cells to target and eliminate undifferentiated senescent cells, thus contributing to endometrial remodeling and rejuvenation (Brighton, Elife 6: e31274, 2017). Through a similar mechanism, in liver fibrosis, senescent hepatic satellite cells expressing p53 polarize resident Kupfer macrophages and newly infiltrating macrophages towards a pro-inflammatory M1 phenotype, exhibiting senescent activity. F4 / 80+ macrophages have been shown to play a crucial role in clearing senescent cells from the mouse uterus to maintain postpartum uterine function.

[0302] Senescent cells primarily recruit NK cells by upregulating NKG2D ligands (expressed on NK cells), chemokines, and other SASP factors. In vivo models of liver fibrosis have shown that activated NK cells effectively clear senescent cells (Krizhanovsky, Cell 134(4): 657-667, 2008). Studies have described various models to investigate aging, including liver fibrosis (Krizhanovsky, Cell 134(4): 657-667, 2008), osteoarthritis (Xu, J Gerontol A Biol Sci Med Sci 72(6): 780-785, 2017), and Parkinson's disease (Chinta, Cell Reports 22(4): 930-940, 2018). Animal models used to study senescent cells are described in the following literature: Krizhanovsky, Cell 134(4): 657-667, 2008; Baker, Nature 479(7372): 232-236, 2011; Farr, Nat Med 23(9): 1072-1079, 2017; Bourgeois, FEBS Lett 592(12): 2083-2097, 2018; Xu, Nat Med 24(8): 1246-1256, 2018.

[0303] Other treatments

[0304] Some embodiments of any of the methods described herein may further include administering a therapeutically effective amount of one or more additional therapeutic agents to a subject (e.g., any subject described herein). One or more additional therapeutic agents may be administered to the subject substantially simultaneously with the multi-chain chimeric peptide (e.g., any multi-chain chimeric peptide described herein). In some embodiments, one or more additional therapeutic agents may be administered to the subject prior to the administration of the multi-chain chimeric peptide (e.g., any multi-chain chimeric peptide described herein). In some embodiments, one or more additional therapeutic agents may be administered to the subject after the administration of the multi-chain chimeric peptide (e.g., any multi-chain chimeric peptide described herein).

[0305] Other non-limiting examples of therapeutic agents include: anti-inflammatory agents, anticancer drugs, receptor activators, immune checkpoint inhibitors, agents for blocking HLA-specific inhibitory receptors, glycogen synthase kinase (GSK) 3 inhibitors, antibodies, and ex vivo activated immune cells (e.g., CAR-T cells or CAR NK cells).

[0306] Non-limiting examples of anticancer drugs include antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, fluxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, 6-thioguanine, cladribine, nelarabine, pentostatin, or pemetrexed), alkaloids (e.g., vinblastine, vincristine, vindesine, camptothecin, 9-methoxycamptothecin, coronaridine, paclitaxel, naucleaoral, and diprenylated indole alkaloids). alkaloid, montamine, schischkiniin, protoberberine, berberine, sanguinarine, chelerythrine, chelidonine, liriodenine, clivorine, β-carboline, antholine, tylophorine, cryptolepi Neocryptolepine, corynoline, sampangine, carbazole, crinamine, montanine, ellipticine, paclitaxel, docetaxel, etoposide, tenisopide, irinotecan, topotecan, or acridinone alkaloids), proteasome inhibitors (e.g.,Lactacystin, disulfiram, epigallocatechin-3-gallate, marizomib (salinosporamide A), oprozomib (ONX-0912), delanzomib (CEP-18770), epoxomicin, MG132, β-hydroxyβ-methylbutyrate, bortezomib, carfilzomib, or ixazomib, and antitumor antibiotics (e.g., doxorubicin) doxorubicin, daunorubicin, epirubicin, mitoxantrone, idarubicin, actinomycin, plicamycin, mitomycin, or bleomycin, histone deacetylase inhibitors (e.g., vorinostat, panobinostat, belinostat, givinostat, abexinstat) abexinostat, depsipeptide, entinostat, phenylbutyrate, valproic acid, trichostatin A, dacinostat, mocetinostat, pracinostat, nicotinamide, cambinol, tenovin 1, tenovin 6, sirtinol, ricolinostat, tefinostat, kevelin rin), quisinostat, resminostat, tacedinaline, chidamide, or selisistat, tyrosine kinase inhibitors (e.g., axitinib, dasatinib, encorafinib, erlotinib, imatinib, nilotinib, pazopanib, and sunitinib), and chemotherapy agents (e.g.,All-trans retinoic acid, azacitidine, azathioprine, deoxyfluorouridine, epothilone, hydroxyurea, imatinib, teniposide, thioguanine, valrubicin, vemurafenib, and lenalidomide. Other examples of chemotherapy agents include alkylating agents, such as dichloromethyldiethylamine, cyclophosphamide, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, altretamine, procarbazine, dacarbazine, temozolomide, carmustine, lumustine, streptozocin, carboplatin, cisplatin, and oxaliplatin.

[0307] Non-limiting examples of receptor activators include any activator of a receptor that activates and enhances the cytotoxicity of NK cells, including anti-CD16 antibodies (e.g., anti-CD16 / CD30 bispecific monoclonal antibody (BiMAb)) and Fc-based fusion proteins. Non-limiting examples of checkpoint inhibitors include anti-PD-1 antibodies (e.g., MEDI0680), anti-PD-L1 antibodies (e.g., BCD-135, BGB-A333, CBT-502, CK-301, CS1001, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316, anti-PD-L1 / CTLA-4 bispecific antibody KN046, and anti-PD-L1 / TGFβRII fusion proteins). Synthetic protein M7824, anti-PD-L1 / TIM-3 bispecific antibody LY3415244, atezolizumab or avelumab, anti-TIM3 antibodies (e.g., TSR-022, Sym023 or MBG453), and anti-CTLA-4 antibodies (e.g., AAGEN1884, MK-1308 or anti-CTLA-4 / OX40 bispecific antibody ATOR-1015). Non-limiting examples of agents for blocking HLA-specific inhibitory receptors include monalizumab (e.g., an anti-HLA-E NKG2A inhibitory receptor monoclonal antibody). Non-limiting examples of GSK3 inhibitors include tideglusib or CHIR99021. Non-limiting examples of antibodies that can be used as additional therapeutic agents include anti-CD26 antibodies (e.g., YS110), anti-CD36 antibodies, and any other antibodies or antibody constructs that can bind to and activate an Fc receptor (e.g., CD16) on NK cells. In some embodiments, the additional therapeutic agent may be insulin or metformin.

[0308] Non-restricted examples of in vitro activated immune cells include regulatory T cells, CAR-regulatory T cells, NK cells, CAR-NK cells, cytotoxic T cells, and CAR-cytotoxic T cells. Example

[0309] The invention is further illustrated in the following examples, which do not limit the scope of the invention as described in the claims.

[0310] Example 1. Construction and characterization of exemplary multi-chain chimeric peptides

[0311] Two multi-chain chimeric peptides were generated and their properties were evaluated. Each of the two multi-chain chimeric peptides comprises a first chimeric peptide containing a soluble tissue factor domain covalently linked to a first domain of an affinity domain pair. The second chimeric peptide in each of the two multi-chain chimeric peptides contains a second domain of the affinity domain pair and a second target-binding domain. Activity data for various chimeric peptides are illustrated, for example, in U.S. Applications 17 / 819,240 and 16 / 555,689, each of which is incorporated herein by reference in its entirety.

[0312] Description of the construction logic of multi-chain chimeric peptides

[0313] Tissue factor (TF) is a stable transmembrane protein containing 236 amino acid residues. The truncated recombinant 219-amino acid extracellular domain of TF is soluble and is known to be highly expressed in bacterial or mammalian cells. Not wishing to be bound by any particular theory, the applicant hypothesizes that 219-aa TF could serve as a linker to form unique multi-chain chimeric polypeptides.

[0314] The first chimeric peptides containing a soluble tissue factor domain were produced at high levels by CHO cells grown in fermentation broth. These first chimeric peptides were purified by anti-tissue factor monoclonal antibody (mAb) conjugated to a solid matrix. Notably, tissue factor contains binding sites for both FVIIa and FX. When tissue factor is not anchored to a phospholipid bilayer, the catalytic activity of the tissue factor-FVIIa complex for FX is approximately 1 / 1,000,000. Therefore, not wanting to be bound by any particular theory, the applicant hypothesizes that using the 219-aa extracellular domain of tissue factor, which lacks a transmembrane structure, during the construction of the first chimeric peptides could eliminate the procoagulant activity of tissue factor in the first chimeric peptides. To further reduce or eliminate the procoagulant activity of 219-aa tissue factor, selective mutations can be made in tissue factor, particularly at the seven amino acid residues known to contribute to the binding energy of the FVIIa binding site.

[0315] Characterization of the binding interactions of the described chimeric peptides

[0316] To determine whether the first and second chimeric peptides bind to each other to form a multi-chain chimeric peptide, in vitro binding assays were performed. To determine whether the first chimeric peptide containing a soluble tissue factor domain was recognized and bound by anti-TF mAb, in vitro binding assays were performed. Notably, the data indicate that the mutated tissue factor protein was still recognized and selectively bound by anti-TF mAbs known to bind to the FX binding site on tissue factor. To determine the first chimeric peptide containing a soluble tissue factor domain covalently linked to scFv or cytokines (see [link to relevant documentation]), further assays were performed. Figure 1 and Figure 2 Whether the scFv or cytokine possesses functionality was determined by an in vitro binding assay. Data from the aforementioned assay were consistent with purified first chimeric peptides exhibiting the expected biological activity (e.g., scFv selectively binds to the expected target antigen, or cytokines selectively bind to the expected receptor or binding protein).

[0317] Furthermore, experiments using two multi-chain chimeric peptides, comprising a first chimeric peptide and a second chimeric peptide that bind to each other, demonstrated the expected target-binding activity (e.g., the multi-chain chimeric peptides specifically bind to targets specifically recognized by the first target-binding domain and targets specifically recognized by the second target-binding domain).

[0318] Based on the foregoing results, the applicant concludes that the soluble tissue factor linker provides or enables the appropriate display of peptides encoding scFv, interleukins, cytokines, interleukin receptors, or cytokine receptors in three-dimensional space relative to the soluble tissue factor domain and relative to each other, such that each peptide retains its intended biological properties and activity.

[0319] When both the first and second chimeric peptides are co-expressed, the heterodimeric complex is secreted into the fermentation broth at high levels. The complex was easily purified using affinity chromatography by capturing and conjugating an anti-TF mAb to a solid matrix. The first and second target-binding domains of these multi-chain chimeric peptides retained their expected biological activities, as determined by in vitro binding assays. Therefore, the assembly of multi-chain chimeric peptides provides appropriate spatial display and domain folding for biological activity. Importantly, the spatial arrangement of the multi-chain chimeric peptides does not interfere with the FX binding sites on tissue factor, enabling affinity purification using anti-TF mAbs.

[0320] Characterization of the stability of the described chimeric peptide

[0321] Both purified multi-chain chimeric peptides were stable. These peptides maintained their structural integrity and full biological activity after incubation in human serum at 37°C for 72 hours.

[0322] Characterization of the described chimeric polypeptide aggregation tendency

[0323] The two purified multi-chain chimeric peptides developed did not form aggregates when stored in PBS at 4°C.

[0324] Characterization of the viscosity of the described chimeric polypeptide

[0325] There is no viscosity issue when multi-chain chimeric peptides are prepared in PBS at concentrations up to 50 mg / mL.

[0326] Other applications of multi-chain chimeric peptide platforms

[0327] Data from these studies demonstrate that molecules capable of fusing with antibody-derived target-binding domains can be formed in any of the forms described herein using the platform technologies described herein, including but not limited to adhesion molecules, receptors, cytokines, ligands, and chemokines. With appropriate target-binding domains, the resulting multi-chain chimeric peptides can promote the conjugation and mediating of various immune effector cells against target cells, including cancer cells, virus-infected cells, or senescent cells. Other domains in the multi-chain chimeric peptides stimulate, activate, and attract the immune system to enhance the cytotoxicity of effector cells against targeted cells.

[0328] Example 2: Generation and characterization of TGFRt15-TGFR fusion protein

[0329] The resulting fusion protein complex contains TGFβ receptor II / IL-15RαSu and TGFβ receptor II / TF / IL-15 fusion proteins ( Figure 3 and Figure 4 The human TGFβ receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were obtained from the UniProt website, and DNA of these sequences was synthesized by Genewiz. Specifically, a construct was formed in which the two TGFβ receptor II sequences were ligated to a G4S(3) linker to generate a single-stranded form of TGFβ receptor II, and then directly ligated to the N-terminal coding region of tissue factor 219, followed by ligation to the N-terminal coding region of IL-15.

[0330] The nucleic acid and protein sequences of such a construct are shown below, which contains two TGFβ receptor IIs linked to the N-terminus of tissue factor 219, and then linked to the N-terminus of IL-15.

[0331] The nucleic acid sequences (including the signal peptide sequence) of the two TGFβ receptor II / TF / IL-15 constructs are as follows (SEQ ID NO:35):

[0332] (Signal peptide)

[0333] ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC

[0334] (Two-person TGFβ receptor II fragment)

[0335] ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

[0336] (Human Tissue Factor 219)

[0337] AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

[0338] (Human IL-15)

[0339] AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

[0340] The amino acid sequence (including the leader sequence) of the TGFβ receptor II / TF / IL-15 fusion protein is as follows (SEQ ID NO:36):

[0341] (Signal peptide)

[0342] MKWVTFISLLFLFSSAYS

[0343] (Human TGFβ receptor II)

[0344] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGG GGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0345] (Human tissue factor 219)

[0346] SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0347] (Human IL-15)

[0348] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0349] The construct was also prepared by directly linking two TGFβ receptor IIs to an IL-15RαSu chain synthesized by Genewiz. The nucleic acid and protein sequences of the construct containing the TGFβ receptor II linked to the N-terminus of IL-15RαSu are shown below.

[0350] The nucleic acid sequence (including the signal peptide sequence) of the TGFβ receptor II / IL-15 RαSu construct is as follows (SEQ ID NO:37):

[0351] (Signal peptide)

[0352] ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCCAGCGCCTACTCC

[0353] (Two TGFβ receptor II fragments)

[0354] ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

[0355] (Human IL-15R α sushi domain)

[0356] ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

[0357] The amino acid sequences (including the signal peptide sequence) of the two TGFβ receptor II / IL-15RαSu constructs are as follows (SEQ ID NO:38):

[0358] (Signal peptide)

[0359] MKWVTFISLLFLFSSAYS

[0360] (Extracellular domain of TGFβ receptor II in two individuals)

[0361] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGG GGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0362] (Human IL-15R α sushi domain)

[0363] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0364] In some cases, the precursor peptide is cleaved from the whole polypeptide to produce a soluble or secreted mature form.

[0365] The TGFβR / IL-15RαSu and TGFβR / TF / IL-15 constructs were cloned into a modified retroviral expression vector, as previously described (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther [Human Gene Therapy] 2005; 16:457-72), and the expression vector was transfected into CHO-K1 cells. Co-expression of the two constructs in CHO-K1 cells allowed for the formation and secretion of a soluble TGFβR / TF / IL-15:TGFβR / IL-15RαSu protein complex (referred to as TGFRt15-TGFR), which could be purified by anti-TF IgG1 affinity and other chromatographic methods.

[0366] Example 3: TGFRt15 -TGFR generation

[0367] A fusion protein complex containing TGFR / IL15RαSu and TGFR / TF / IL-15D8N fusion proteins was generated. Human TGF-β receptor (TGFR), IL-15α receptor sushi domain (IL15RaSu), tissue factor (TF), and IL-15 with the D8N mutant (IL15D8N) sequences were obtained from GenBank, and DNA fragments of these sequences were synthesized by Genewiz. Specifically, a construct was prepared by linking the TGFR sequence to the N-terminal coding region of IL15RaSu, and then linking the TGFR sequence to the N-terminus of tissue factor 219, followed by linking it to the N-terminal coding region of IL-15D8N.

[0368] The nucleic acid sequence (including the signal peptide sequence) of the TGFR / IL15RαSu construct is as follows (SEQ ID NO:39):

[0369] (Signal peptide)

[0370] ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCCAGCGCCTACTCC

[0371] (Single-chain human TGF-β receptor II homodimer)

[0372] ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

[0373] (Sushi domain of the IL15 receptor α-chain)

[0374] ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

[0375] The nucleic acid sequence (including the signal peptide sequence) of the TGFR / TF / IL15D8N construct is as follows (SEQ ID NO:40):

[0376] (Signal peptide)

[0377] ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCC

[0378] (Single-chain human TGF-β receptor II homodimer)

[0379] ATCCCACCGCACGTTCAGAAGTCGGTGAATAACGACATGATAGTCACTGACAACAACGGTGCAGTCAAGTTTCCACAACTGTGTAAATTTTGTGATGTGAGATTTTCCACCTGTGACAACCAGAAATCCTGCATGAGCAACTGCAGCATCACCTCCATCTGTGAGAAGCCACAGGAAGTCTGTGTGGCTGTATGGAGAAAGAATGACGAGAACATAACACTAGAGACAGTTTGCCATGACCCCAAGCTCCCCTACCATGACTTTATTCTGGAAGATGCTGCTTCTCCAAAGTGCATTATGAAGGAAAAAAAAAAGCCTGGTGAGACTTTCTTCATGTGTTCCTGTAGCTCTGATGAGTGCAATGACAACATCATCTTCTCAGAAGAATATAACACCAGCAATCCTGACGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC

[0380] (Human Tissue Factor 219)

[0381] TCAGGCACTACAAATACTGTGGCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTGGAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGCACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACAGAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTACTTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGTTCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTACCTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTGGGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGAAGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTAATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACAGCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAAAACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAACCGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGGGAATTCAGAGAA

[0382] (Human IL-15 D8N)

[0383] AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCA AGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT

[0384] The amino acid sequence (including the signal peptide sequence) of the TGFR / IL15RaSu fusion protein is as follows (SEQ ID NO:41):

[0385] (Signal peptide)

[0386] MKWVTFISLLFLFSSAYS

[0387] (Single-chain human TGF-β receptor II homodimer)

[0388] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGG GGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0389] (human IL-15 receptor α-sushi domain)

[0390] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0391] The amino acid sequence (including the signal peptide sequence) of the TGFR / TF / IL15D8N fusion protein is as follows (SEQ ID NO:42):

[0392] (Signal peptide)

[0393] MGVKVLFALICIAVAEA

[0394] (Single-chain human TGF-β receptor II homodimer)

[0395] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGG GGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0396] (Organization factor)

[0397] SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0398] (IL-15D8N)

[0399] NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0400] The TGFR / IL15RαSu and TGFR / TF / IL-15D8N constructs were cloned into a modified retroviral expression vector, as previously described (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al.). The expression vector was transfected into CHO-K1 cells. Co-expression of these two constructs in CHO-K1 cells allowed for the formation and secretion of a soluble TGFR / IL15RαSu-TGFR / TF / IL-15D8N protein complex (referred to as TGFRt15). The protein complex (-TGFR) can be purified using anti-TF antibody affinity.

[0401] Example 4: TGFRt15-TGFR (HCW9218), HCW9228 and TGFR t15-TGFR TGFβ1 junction of (HCW9238) combine

[0402] TGFRt15-TGFR (HCW9218) (10 µg / mL), HCW9228 (10 µg / mL) and TGFR t15-TGFR (HCW9238) (10 µg / mL) was added to 96-well plates coated with anti-TF IgG1 and incubated at room temperature (RT). The plates were washed and blocked with blocking buffer of 1% BSA in PBS. The plates were then washed again, and TGF-β1 (BioLegend) was titrated from 4000 pM down to 31 pM into the plates, which were then incubated at RT. The plates were then washed again, and 200 ng / mL biotinylated anti-TGFβRI antibody (BAF240, R&D Systems) was added to each well, which was then incubated at RT. Next, the plates were washed again, and 0.2 µg / mL HRP-SA (Jackson ImmunoResearch) was added to each well and incubated at RT, followed by incubation with ABTS at RT. The absorbance was read at 405 nm. Figure 5 As shown, TGFβ1 binding was detected by biotinylated anti-TGFβ1. The results indicated that, compared to TGFRt15-TGFR (HCW9218) and HCW9228, TGFR... t15-TGFR (HCW9238) exhibits low binding activity to TGF-β1.

[0403] Example 5: TGFRt15-TGFR (HCW9218), HCW9228 and TGFR t15-TGFR LAP binding of (HCW9238)

[0404] TGFRt15-TGFR (HCW9218) (10 µg / mL), HCW9228 (10 µg / mL) and TGFR t15-TGFR (HCW9238) (10 µg / mL) was added to a 96-well plate coated with anti-TF IgG1 and incubated at room temperature (RT). The plate was washed and blocked with blocking buffer of 1% BSA in PBS. Then, the plate was washed, and LAP (Research Systems) was titrated from 4000 pM down to 31 pM into the plate, and the plate was incubated at RT. Then, the plate was washed, and 100 ng / mL biotinylated anti-LAP antibody (BAM2462, Research Systems) was added to the wells, and the plate was incubated at RT. Next, the plate was washed, and 0.2 µg / mL HRP-SA (Jackson Immunological Research) was added to each well and incubated at RT, followed by incubation at RT with ABTS. The absorbance was read at 405 nm. Figure 6 As shown, LAP binding was detected using anti-human LAP antibodies. The results indicated that, compared to TGFRt15-TGFR (HCW9218) and HCW9228, TGFR... t15-TGFR (HCW9238) exhibits low binding activity to LAP.

[0405] Example 6: Characterization of HCW9238

[0406] In the presence of TGF-β1, TGF-β-responsive HKT-TGF-β cells were compared with TGFRt15-TGFR (HCW9218) (10 µg / mL), HCW9228 (10 µg / mL) and TGFR. t15-TGFR (HCW9238) was incubated together with test medium (10% HI-FCS). After incubation, the culture supernatant was mixed with QUANTI-Blue (InvivoGen) and incubated for 1-3 hours. OD620 values ​​were measured using a microplate reader. Blocking activity was calculated using GraphPad Prism 7.04. Figure 7A The results showed that, compared with TGFRt15-TGFR (HCW9218) and HCW9228, TGFR t15-TGFR (HCW9238) exhibits TGF-β1 antagonist activity.

[0407] IL-15-responsive CTLL-2 cells were used to compare TGFRt15-TGFR (HCW9218) (10 µg / mL), HCW9228 (10 µg / mL), and TGFR. t15-TGFR The bioactivity of (HCW9238) was assessed. CTLL-2 cells were cultured in RPMI-10 medium, and PrestoBlue (Fisher Scientific) was added to the culture medium on the last day. Cell proliferation was then determined using a microplate reader (OD570-600 value). Figure 7B The results show that, compared to HCW9228, TGFR t15-TGFR (HCW9238) and TGFRt15-TGFR (HCW9218) exhibited similar IL-15 activity and greater IL-15 activity.

[0408] Example 7: IL-15 activity of HCW9238 and 32Dβ and CTLL2 cells

[0409] To evaluate TGFR t15-TGFR The activity of IL-15 in (HCW9238) was evaluated using 32Dβ cells expressing IL2Rβ and the common γ chain, and their effects on promoting cell proliferation were assessed to reduce TGFR t15-TGFR The IL-15 activity of (HCW9238) was compared with that of TGFRt15-TGFR (HCW9218). IL-15-dependent 32Dβ cells were washed with IMDM-10% FBS and subjected to 2 × 10⁻⁶ ppm. 4 Cells were seeded per well. On the last day of culture, PrestoBlue (Feishier Technology Co., Ltd.) was added, and cell proliferation was determined using a microplate reader (OD570-600 value). Figure 8A ).

[0410] Using CTLL-2 cells, the differences between TGFRt15-TGFR (HCW9218) (10 µg / mL) and TGFR were compared. t15-TGFR The activity of IL-15 in (HCW9238) was assessed. CTLL-2 cells were cultured in RPMI-10, and PrestoBlue (Feisil Technology Co., Ltd.) was added to the culture medium on the last day. Cell proliferation was then determined using a microplate reader (OD570-600 value). Figure 8B ).

[0411] The results showed that TGFR t15-TGFR (HCW9238) and TGFRt15-TGFR (HCW9218) showed similar IL-15 activity against CTLL-2 cells, and TGFRt15-TGFR (HCW9218) showed better activity against 32Dβ cells than TGFR. t15-TGFR (HCW9238) has greater IL-15 activity.

[0412] Example 8: Comparison of clearance of HCW9218, -28, and -38 from mouse blood

[0413] Evaluation of TGFRt15-TGFR (HCW9218), HCW9228, and TGFR in wild-type female C57BL / 6 mice t15-TGFR Pharmacokinetics of the molecule (HCW9238). Using TGFRt15-TGFR (HCW9218), HCW9228, and TGFR... t15-TGFR (HCW9238) Subcutaneous treatment of mice. On day 4, blood was collected from the tail vein of mice and serum was prepared. TGFRt15-TGFR (HCW9218), HCW9228, and TGFR in mouse serum were determined by ELISA. t15-TGFR (HCW9238) Concentration (Capture: Anti-human tissue factor antibody; Detection: Biotinylated anti-human TGFβ receptor antibody, followed by peroxidase-conjugated streptavidin and ABTS substrate) Figure 9 ). With HCW9228 and TGFR t15-TGFR Compared to (HCW9238), a higher TGFR was detected on day 4 post-treatment. t15-TGFR (HCW9238) serum concentration, indicating TGFR t15-TGFR (HCW9238) has a longer half-life in vivo.

[0414] Example 9: Comparison of HCW9218, -28, and -38 in immune stimulation

[0415] Mouse spleen cells were prepared to evaluate TGFRt15-TGFR (HCW9218), HCW9228 and TGFR. t15-TGFR (HCW9238) immunostimulatory activity over time in mice. For example... Figure 10 As shown, using TGFR t15-TGFR Spleen weight increased in mice treated with (HCW9238) at 96 hours post-treatment. Furthermore, the spleen weight of control-treated mice and mice treated with TGFRt15-TGFR (HCW9218), HCW9228, and TGFR was evaluated. t15-TGFR CD4 present in the spleen of (HCW9238) treated mice + T cells, CD8 + The percentage of T cells and NK cells. Compared to control mice, TGFR t15-TGFR (HCW9238) administration induced CD8+ in the spleen of mice. + The percentages of T cells and NK cells increased.

[0416] In addition, single doses of TGFRt15-TGFR (HCW9218), HCW9228 and TGFR t15-TGFR Following (HCW9238), the dynamic response of immune cells based on the expression of Ki67 (proliferation), KLRG1 and CD25 (activation), and granzyme B (cytotoxic potential) was evaluated in spleen cells isolated from mice. Figure 10 When compared with control mice, TGFR t15-TGFR (HCW9238) administration led to CD8+ oxidase in spleen cells. + Increased activation, proliferation, and cytotoxic potential of T cells and NK cells.

[0417] Example 10: Immune infiltration kinetics of HCW9328 in melanoma-bearing mice

[0418] In preclinical models, HCW9328 expands antigen-experienced CD8+ T cells in blood, draining lymph nodes, and tumors. Six-week-old C57Bl6 / j cells were subcutaneously injected with 0.5 × 10⁻⁶ cells. 6 Mice were treated with B16F10 melanoma tumor cells. When tumors were visible, mice received either HCW9218 (3 mg / kg) or HCW9328 (3 mg / kg) on ​​day 8 post-tumor implantation. Some mice received saline as a control. Mice were sacrificed at 24 h, 48 h, 72 h, and 120 h post-treatment. Tumors, draining inguinal lymph nodes (dLNs), and peripheral blood were processed for single-cell suspension and stained with commercially available fluorescently labeled antibodies. Population absolute counts were determined by flow cytometry using absolute counting beads. Samples were acquired using a BD Celesta flow cytometer.

[0419] As shown in Figure 11, HCW9328 amplified antigen-experienced (CD44+) CD8+ T cells in the blood at 24 h, 48 h, 72 h, and 120 h post-treatment. In the dLN, amplification was observed at 48 h, 72 h, and 120 h post-treatment. In the tumor, CD44+CD8 T cells amplified at 72 h post-treatment. HCW9218 also followed similar pharmacodynamics to HCW9238. In the blood, proliferating Ki67+ CD44+ CD8 T cells were observed at all time points analyzed after HCW9238 treatment. In the dLN, CD44+ CD8 T cell proliferation was observed from 48 h to 72 h, followed by a decrease at 120 h. In the tumor, CD44+ Ki67+ CD8 T cells were observed at 48 h and 72 h post-treatment. Compared with mice treated with HCW9238, mice treated with HCW9218 showed proliferating CD8 T cells in the tumor at 24 h.

[0420] Figure 12 illustrates the in vivo activity of HCW9238 in tumor-bearing mice. HCW9218 was used as a control for these experiments. HCW9238 showed a higher proliferative (Ki67) subset in all tissues analyzed. HCW9238 expanded antigen-experienced CD8+ T cells and Tpex cells in both the dLN and tumors, and expanded the Tex population in tumors. The data presented in this paper indicate that the IL-15-stimulatory activity of HCW9238 can activate and expand CD8 T cell subsets, such as Tpex, which are cells that enter the tumor from the dLN and have potent antitumor activity. Other embodiments

[0421] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are all within the scope of the following claims.

Claims

1. A multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (a) A first chimeric polypeptide comprising: (i) A first target-binding domain, the first target-binding domain comprising: A first sequence that is at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine, and the amino acid at position 119 of SEQ ID NO:2 is alanine; and The second sequence is at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine; (ii) a soluble tissue factor domain comprising at least 90% of the sequence identical to SEQ ID NO:1; and (iii) The first domain of the affinity pair, the first domain containing at least 90% identical sequence to SEQ ID NO:13; and (b) A second chimeric polypeptide comprising: (i) a second domain of the affinity pair, the second domain containing at least 90% identical sequence to SEQ ID NO:11; and (ii) a second target-binding domain, the second target-binding domain comprising: A first sequence that is at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine, and the amino acid at position 119 of SEQ ID NO:2 is alanine; and The second sequence is at least 90% identical to SEQ ID NO:2, wherein the amino acid at position 32 of SEQ ID NO:2 is asparagine and the amino acid at position 119 of SEQ ID NO:2 is alanine; in: The first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first and second domains of the affinity pair.

2. The multi-chain chimeric polypeptide of claim 1, wherein the first target-binding domain comprises at least 80% of the same sequence as SEQ ID NO:

4.

3. The multi-chain chimeric polypeptide of claim 1, wherein the first chimeric polypeptide comprises at least 80% of the same sequence as SEQ ID NO:

6.

4. The multi-chain chimeric polypeptide of claim 1, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

7.

5. The multi-chain chimeric polypeptide of claim 1, wherein the second target-binding domain comprises at least 80% of the same sequence as SEQ ID NO:

4.

6. The multi-chain chimeric polypeptide of claim 1, wherein the second chimeric polypeptide comprises at least 80% of the same sequence as SEQ ID NO:

5.

7. The multi-chain chimeric polypeptide of claim 1, wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO:

8.

8. A pharmaceutical composition comprising any one of the multi-chain chimeric polypeptides as described in claims 1-7.

9. A kit comprising at least one dose of the composition as claimed in claim 8.

10. A method for stimulating and / or inducing or increasing the proliferation of immune cells, the method comprising contacting the immune cells with an effective amount of any one of the multi-chain chimeric polypeptides as described in claims 1-7.

11. A method for inducing immune cells to differentiate into memory or memory-like immune cells, the method comprising contacting the immune cells with an effective amount of any one of the multi-chain chimeric polypeptides as described in claims 1-7.

12. The method of claim 10 or 11, wherein the immune cells are contacted in vitro or in vivo.

13. The method of any one of claims 10-12, wherein the immune cell is selected from the group consisting of: immature thymocytes, peripheral blood lymphocytes, primary T cells, pluripotent Th cell precursors, lymphoid progenitor cells, Treg cells, Th17 cells, Th22 cells, Th9 cells, Th2 cells, Th1 cells, Th3 cells, γδ T cells, αβ T cells, tumor-infiltrating T cells, CD8+ T cells, CD4+ T cells, natural killer T cells, mast cells, macrophages, neutrophils, dendritic cells, basophils, eosinophils, and natural killer cells.

14. The method of any one of claims 10-13, wherein the immune cell has been previously genetically modified to express a chimeric antigen receptor or a recombinant T-cell receptor.

15. A method for killing cancer cells, infected cells, or senescent cells in a subject in need, the method comprising administering to the subject a therapeutically effective amount of any one of the multi-chain chimeric polypeptides as described in claims 1-7.

16. The method of claim 15, wherein the subject has been identified or diagnosed with cancer, an age-related disease or condition, or an infectious disease.

17. A composition for treating a subject in need, the composition comprising any one of the multi-chain chimeric polypeptides as described in claims 1-7.

18. A nucleic acid encoding any one of the multi-chain chimeric polypeptides as described in any one of claims 1-7.

19. A vector comprising the nucleic acid as described in claim 18.

20. A cell comprising the nucleic acid as described in claim 18.

21. A cell comprising the carrier as described in claim 19.

22. A method for generating a multi-chain chimeric polypeptide, the method comprising: The cells as described in claim 20 are cultured in a culture medium under conditions sufficient to induce the production of the multi-chain chimeric polypeptide. as well as The multi-chain chimeric polypeptide was recovered from the cell and / or the culture medium.

23. A multi-chain chimeric polypeptide, which is generated by the method of claim 22.

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