Multi-lock chimeric polypeptides and their use

Abstract

The present disclosure relates to methods for stimulating NK cells, methods for inducing or increasing NK cell proliferation, methods for inducing NK cell differentiation, and methods for using activated NK cells to treat a subject in need thereof, including the use of a first multi-chain chimeric polypeptide and a second multi-chain chimeric polypeptide.

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 864,996, filed on June 21, 2019, the entire content of which is incorporated herein by reference.

[0002] Technical Field The present disclosure relates to the field of biotechnology, and more specifically, to the use of a combination of two multichain chimeric polypeptides, each having a linker domain disposed between two target - binding domains.

Background Art

[0003] Background Adoptive immunotherapy or cell therapy requires culturing of immune cells obtained from a subject in vivo (and optionally genetic engineering of immune cells to express a T - cell receptor or chimeric antigen receptor) before administering them back to the subject. To provide a therapeutic effect to the subject, a sufficient number of immune cells are required. In many instances, immune cells obtained from a subject must be cultured for more than three weeks, after which a therapeutically effective number of immune cells can be obtained. In addition, many methods of culturing immune cells obtained from a subject in vitro require a feeder cell layer, which requires subsequent purification or isolation of the immune cells before administering them back to the subject.

Summary of the Invention

[0004] Summary The present invention is based on the discovery that a specific two - step method of contacting NK cells results in significant in vitro proliferation of NK cells and highly activated NK cells that grow with long persistence and high cytotoxicity against diseased cells in a recipient host.

[0005] In one aspect, provided herein is a method for promoting the activation and proliferation of natural killer cells, the method comprising: (a) contacting natural killer cells with an effective amount of a first multichain chimeric polypeptide in a liquid culture medium for a first period under conditions that allow for the differentiation of natural killer cells; and (b) contacting natural killer cells with an effective amount of (i) a second multichain chimeric polypeptide and (ii) an IgG1 antibody construct in a liquid culture medium for a second period under conditions that allow for the activation and proliferation of natural killer cells, wherein the first multichain chimeric polypeptide comprises a first chimeric polypeptide comprising a first target binding domain, a soluble tissue factor domain, and a first domain of an affinity domain pair, and a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide within the first multichain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the first multichain chimeric polypeptide, and the first target binding domain and the second target binding domain within the first multichain chimeric polypeptide each independently and specifically bind to an IL-18 receptor or an IL-12 receptor, the second multichain chimeric polypeptide comprises a first chimeric polypeptide comprising a first target binding domain, a linker domain, and a first domain of an affinity domain pair, and a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide within the second multichain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the second multichain chimeric polypeptide, and the first target binding domain and the second target binding domain within the second multichain chimeric polypeptide each independently and specifically bind to an IL-7 receptor or an IL-21 receptor, and the IgG1 antibody construct comprises at least one antigen binding domain that specifically binds to the linker domain.

[0006] In some embodiments, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide in the first multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide. In some embodiments, the soluble tissue factor domain and the first domain of the affinity domain pair within the first chimeric polypeptide in the first multi-chain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair within the first chimeric polypeptide. In some embodiments, the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide in the first multi-chain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide in the first multi-chain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 9.In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain does not include one or more of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind to factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X to factor Xa.In some embodiments, the first multi-chain chimeric polypeptide does not stimulate coagulation in mammals. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide within the first multi-chain chimeric polypeptide further comprises a signal sequence at its N-terminus. In some embodiments, the signal sequence comprises SEQ ID NO: 28. In some embodiments, the signal sequence is SEQ ID NO: 28. In some embodiments, the affinity domain pair within the first multi-chain chimeric polypeptide is the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. In some embodiments, soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises SEQ ID NO: 26. In some embodiments, the sushi domain of IL15Rα comprises the sushi domain derived from human IL15Rα. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 80% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 90% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 95% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα is the mature full-length IL15Rα.

[0007] In some embodiments, the affinity domain pairs within the first multi-chain chimeric polypeptide are selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first target-binding domain and the second target-binding domain within the first multi-chain chimeric polypeptide are each independently soluble IL-15 or soluble IL-18. In some embodiments, the first target-binding domain within the first multi-chain chimeric polypeptide specifically binds to the IL-12 receptor, and the second target-binding domain within the first multi-chain chimeric polypeptide specifically binds to the IL-18 receptor. In some embodiments, the first target-binding domain within the first multi-chain chimeric polypeptide specifically binds to the IL-18 receptor, and the second target-binding domain within the first multi-chain chimeric polypeptide specifically binds to the IL-12 receptor. In some embodiments, the first target-binding domain within the first multi-chain chimeric polypeptide comprises soluble IL-18. In some embodiments, the soluble IL-18 is soluble human IL-18. In some embodiments, the soluble human IL-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 20. In some embodiments, the soluble human IL-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 20. In some embodiments, the soluble human IL-18 comprises a sequence that is at least 95% identical to SEQ ID NO: 20. In some embodiments, the soluble human IL-18 comprises the sequence of SEQ ID NO: 20. In some embodiments, the second target-binding domain within the first multi-chain chimeric polypeptide comprises soluble IL-12. In some embodiments, the soluble IL-12 is soluble human IL-12. In some embodiments, the soluble human IL-12 comprises a soluble human IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence. In some embodiments, the soluble human IL-15 further comprises a linker sequence between the soluble IL-12β (p40) sequence and the soluble human IL-12α (p35) sequence. In some embodiments, the linker sequence comprises SEQ ID NO: 11.In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 14. In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the soluble human IL-12β (p40) sequence comprises SEQ ID NO: 14. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 16. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 16. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the soluble human IL-12α (p35) sequence comprises SEQ ID NO: 16. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide is, comprises a sequence that is at least 80% identical to TIFF0007713393000001.tif66138. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide comprises SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide is, comprises TIFF0007713393000002.tif66138. In some embodiments, the second chimeric polypeptide is, It comprises a sequence that is at least 80% identical to TIFF0007713393000003.tif79138. In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 65. In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 65. In some embodiments, the second chimeric polypeptide comprises SEQ ID NO: 65. In some embodiments, the second chimeric polypeptide is It includes TIFF0007713393000004.tif80138. In some embodiments, the first target binding domain and the linker domain in the first chimeric polypeptide within the second multi-chain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide within the second multi-chain chimeric polypeptide further includes a linker sequence between the first target binding domain and the linker domain in the first chimeric polypeptide of SEQ ID NO: 1. In some embodiments, the linker domain and the first domain of the affinity domain pair are directly adjacent to each other within the first chimeric polypeptide within the second multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide within the second multi-chain chimeric polypeptide further includes a linker sequence between the linker domain in the first chimeric polypeptide and the first domain of the affinity domain pair. In some embodiments, the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide of the second multi-chain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide within the second multi-chain chimeric polypeptide further includes a linker sequence between the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide. In some embodiments, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain includes SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain includes a sequence that is at least 90% identical to SEQ ID NO: 9.In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 10.

[0008] In some embodiments, the soluble human tissue factor domain does not include one or more of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind to factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments, the second multichain chimeric polypeptide does not stimulate coagulation in mammals. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide within the second multichain chimeric polypeptide further includes a signal sequence at its N-terminus. In some embodiments, the signal sequence includes SEQ ID NO: 28. In some embodiments, the signal sequence is SEQ ID NO: 28.In some embodiments, the pair of affinity domains in the second multi-chain chimeric polypeptide is the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. In some embodiments, soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises SEQ ID NO: 26. In some embodiments, the sushi domain of IL15Rα comprises the sushi domain derived from human IL15Rα. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 80% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 90% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 95% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα is the mature full-length IL15Rα. In some embodiments, the pair of affinity domains in the second multi-chain chimeric polypeptide is selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first target binding domain and the second target binding domain in the second multi-chain chimeric polypeptide are each independently soluble IL-7 or soluble IL-21. In some embodiments, the first target binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-21 receptor, and the second target binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-7 receptor.In some embodiments, the first target-binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-7 receptor, and the second target-binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-21 receptor. In some embodiments, the first target-binding domain in the second multi-chain chimeric polypeptide comprises soluble IL-21. In some embodiments, the soluble IL-21 is soluble human IL-21. In some embodiments, the soluble human IL-21 comprises a sequence that is at least 80% identical to SEQ ID NO: 22. In some embodiments, the soluble human IL-21 comprises a sequence that is at least 90% identical to SEQ ID NO: 22. In some embodiments, the soluble human IL-21 comprises a sequence that is at least 95% identical to SEQ ID NO: 22. In some embodiments, the soluble human IL-21 comprises the sequence of SEQ ID NO: 22. In some embodiments, the second target-binding domain comprises soluble IL-7. In some embodiments, the soluble IL-7 is soluble human IL-7. In some embodiments, the soluble human IL-7 comprises a sequence that is at least 80% identical to SEQ ID NO: 23. In some embodiments, the soluble human IL-7 comprises a sequence that is at least 90% identical to SEQ ID NO: 23. In some embodiments, the soluble human IL-7 comprises a sequence that is at least 95% identical to SEQ ID NO: 23. In some embodiments, the soluble human IL-7 comprises the sequence of SEQ ID NO: 23.

[0009] In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide is comprises a sequence that is at least 80% identical to TIFF0007713393000005.tif66138. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide is It includes TIFF0007713393000006.tif66138. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide is, It includes a sequence that is at least 80% identical to TIFF0007713393000007.tif25138. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide includes a sequence that is at least 90% identical to SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide includes a sequence that is at least 95% identical to SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide includes SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide is, It includes TIFF0007713393000008.tif32138. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide is, It includes a sequence that is at least 80% identical to TIFF0007713393000009.tif66138. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide includes a sequence that is at least 90% identical to SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide includes a sequence that is at least 95% identical to SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide includes SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide is, It includes TIFF0007713393000010.tif66138. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide is, TIFF0007713393000011.tif contains a sequence that is at least 80% identical to 31138. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide contains a sequence that is at least 90% identical to SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide contains a sequence that is at least 95% identical to SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide contains SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide is TIFF0007713393000012.tif contains 31138. In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, and both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the linker domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, and one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the linker domain. In some embodiments, the first period is from about 15 minutes to about 4 hours. In some embodiments, the first period is from about 30 minutes to about 3.5 hours. In some embodiments, the first period is from about 1 hour to about 3.5 hours. In some embodiments, the first period is from about 2 hours to about 3.5 hours. In some embodiments, the liquid culture medium of step (a) and / or step (b) is a serum-containing liquid culture medium.

[0010] In some embodiments, the NK cells are about 4.0×10 7 cells / mL to about 1.5×10 8 cells / mL and are present in the liquid culture medium of step (a) at a density of. In some embodiments, the NK cells are about 5.0×10 7 cells / mL to about 2.0×10 8It is present in the liquid culture medium of step (a) at a density of cells / mL. In some embodiments, the second period is from about 1 day to about 30 days. In some embodiments, the second period is from about 7 days to about 30 days. In some embodiments, the second period is from about 14 days to about 21 days. In some embodiments, the liquid culture medium of step (b) contains the second multi-chain chimeric polypeptide and the IgG1 antibody construct at a molar ratio of about 0.5:1 to about 2:1. In some embodiments, the liquid culture medium of step (b) contains the second multi-chain chimeric polypeptide and the IgG1 antibody construct at a molar ratio of about 0.8:1 to about 1.2:1. In some embodiments, NK cells are present in the liquid culture medium of step (b) at about 0.5×10 6 cells / mL to about 2.0×10 6 cells / mL and are maintained at this density. In some embodiments, NK cells are present in the liquid culture medium of step (b) at about 1.0×10 6 cells / mL to about 2.0×10 6It is maintained at a density of cells / mL. In some embodiments, the NK cells are those previously obtained from a subject. In some embodiments, the NK cells are those previously obtained from umbilical cord blood or induced pluripotent stem cells. In some embodiments, the method further comprises obtaining NK cells from a subject prior to step (a). In some embodiments, the NK cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor. In some embodiments, the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells between step (a) and step (b). In some embodiments, the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells prior to step (a). In some embodiments, the method further comprises introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells during step (b). In some embodiments, the method further comprises isolating the NK cells after step (b). In some embodiments, after step (b), the NK cells have an expression or secretion level of one or more proteins that is increased compared to the expression or secretion level of one or more proteins selected from the group consisting of TNF-α, IFN-γ, granzyme A, granzyme B, perforin, 2B4, CD8, CD11a, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CD158a, CD158b, CD158e, CD178, CD226, CD253, NKG2C, NKG2D, LIR-1, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-1, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-α, and IFN-γ before step (a). In some embodiments, the method further comprises administering the NK cells to a subject in need thereof after the contacting step. In some embodiments, the subject is identified or diagnosed as having an age-related disease or condition.In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and kidney dysfunction. In some embodiments, the subject is identified or diagnosed as having cancer. In some embodiments, the cancer is selected from the group consisting of solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B cell neoplasm, multiple myeloma, B cell lymphoma, B cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject is diagnosed or identified as having an infectious disease. In some embodiments, the infectious disease is an infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

[0011] Also provided herein are activated NK cells produced by the method according to any of the above embodiments. Also provided is a pharmaceutical composition comprising the activated NK cells described in the above embodiments. Also provided is a kit comprising the pharmaceutical composition comprising the activated NK cells described in any of the above embodiments. In another aspect, provided herein is a method of killing cancer cells, infected cells, or senescent cells in a subject in need of killing of cancer cells, infected cells, or senescent cells, the method comprising administering to the subject a therapeutically effective amount of the activated NK cells described in any of the above embodiments or the pharmaceutical composition described in any of the above embodiments. In some embodiments, the subject is identified or diagnosed as having cancer. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas, osteosarcomas, B cell neoplasms, multiple myelomas, B cell lymphomas, B cell non-Hodgkin lymphomas, Hodgkin lymphomas, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject is identified or diagnosed as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction.In some embodiments, the subject has been diagnosed or identified as having an infectious disease. In some embodiments, the infectious disease is an infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

[0012] In some embodiments, provided herein is a method of treating a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of the activated NK cells or activated T cells described in any of the above embodiments or the pharmaceutical composition described in any of the above embodiments. In some embodiments, the subject is identified or diagnosed as having cancer. In some embodiments, the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing sarcomas, osteosarcomas, B cell neoplasms, multiple myelomas, B cell lymphomas, B cell non-Hodgkin lymphomas, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject is identified or diagnosed as having an age-related disease or condition. In some embodiments, the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the subject is diagnosed or identified as having an infectious disease.In some embodiments, the infectious disease is an infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus.

[0013] In another aspect, a kit is provided herein, the kit comprising: (a) a first multi-chain chimeric polypeptide comprising a first chimeric polypeptide comprising a first target binding domain, a soluble tissue factor domain, and a first domain of an affinity domain pair, and a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide within the first multi-chain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the first multi-chain chimeric polypeptide, and the first target binding domain and the second target binding domain within the first multi-chain chimeric polypeptide each independently specifically bind to an IL-18 receptor or an IL-12 receptor; a second multi-chain chimeric polypeptide comprising a first chimeric polypeptide comprising a first target binding domain, a linker domain, and a first domain of an affinity domain pair, and a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide within the second multi-chain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the second multi-chain chimeric polypeptide, and the first target binding domain and the second target binding domain within the second multi-chain chimeric polypeptide each independently specifically bind to an IL-7 receptor or an IL-21 receptor; and (c) an IgG1 antibody comprising at least one antigen binding domain that specifically binds to the linker domain. In some embodiments, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide of the first multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide within the first multi-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide. In some embodiments, the soluble tissue factor domain and the first domain of the affinity domain pair within the first chimeric polypeptide of the first multi-chain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide.In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain within the first chimeric polypeptide and the first domain of the affinity domain pair. In some embodiments, the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide within the first multichain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide within the first multichain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 10.In some embodiments, the soluble human tissue factor domain does not include one or more of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain is unable to bind factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments, the first multichain chimeric polypeptide does not stimulate coagulation in mammals. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide within the first multichain chimeric polypeptide further includes a signal sequence at its N-terminus. In some embodiments, the signal sequence includes SEQ ID NO: 28. In some embodiments, the signal sequence is SEQ ID NO: 28.In some embodiments, the affinity domain pair within the first multi-chain chimeric polypeptide is the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. In some embodiments, soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 comprises SEQ ID NO: 26. In some embodiments, the sushi domain of IL15Rα comprises the sushi domain derived from human IL15Rα. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 80% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 90% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 95% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα is the mature full-length IL15Rα. In some embodiments, the affinity domain pair within the first multi-chain chimeric polypeptide is selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first target binding domain and the second target binding domain within the first multi-chain chimeric polypeptide are each independently soluble IL-15 or soluble IL-18. In some embodiments, the first target binding domain within the first multi-chain chimeric polypeptide specifically binds to the IL-12 receptor, and the second target binding domain within the first multi-chain chimeric polypeptide specifically binds to the IL-18 receptor.In some embodiments, the first target binding domain in the first multi-chain chimeric polypeptide specifically binds to the IL-18 receptor, and the second target binding domain in the first multi-chain chimeric polypeptide specifically binds to the IL-12 receptor. In some embodiments, the first target binding domain in the first multi-chain chimeric polypeptide comprises soluble IL-18. In some embodiments, the soluble IL-18 is soluble human IL-18. In some embodiments, the soluble human IL-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 20. In some embodiments, the soluble human IL-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 20. In some embodiments, the soluble human IL-18 comprises a sequence that is at least 95% identical to SEQ ID NO: 20. In some embodiments, the soluble human IL-18 comprises the sequence of SEQ ID NO: 20. In some embodiments, the second target binding domain in the first multi-chain chimeric polypeptide comprises soluble IL-12. In some embodiments, the soluble IL-12 is soluble human IL-12. In some embodiments, the soluble human IL-12 comprises a soluble human IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence. In some embodiments, the soluble human IL-15 further comprises a linker sequence between the soluble IL-12β (p40) sequence and the soluble human IL-12α (p35) sequence. In some embodiments, the linker sequence comprises SEQ ID NO: 11. In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 14. In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the soluble human IL-12β (p40) sequence comprises SEQ ID NO: 14. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 16. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 16.In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the soluble human IL-12α (p35) sequence comprises SEQ ID NO: 16. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide within the first multichain chimeric polypeptide comprises SEQ ID NO: 63. In some embodiments, the first multichain chimeric po. The first chimeric polypeptide within the lipopeptide comprises SEQ ID NO: 64. In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 65. In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 65. In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 65. In some embodiments, the second chimeric polypeptide comprises SEQ ID NO: 65. In some embodiments, the second chimeric polypeptide comprises SEQ ID NO: 66. In some embodiments, the first target binding domain and the linker domain within the first chimeric polypeptide within the second multichain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain within the first chimeric polypeptide. In some embodiments, the linker domain and the first domain of the affinity domain pair are directly adjacent to each other within the first chimeric polypeptide within the second multichain chimeric polypeptide. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide further comprises a linker sequence between the linker domain and the first domain of the affinity domain pair within the first chimeric polypeptide. In some embodiments, the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide within the second multichain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide. In some embodiments, the linker domain is a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5.In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 5. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 9. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 3. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO: 10. In some embodiments, the soluble human tissue factor domain does not include one or more of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein.In some embodiments, the soluble human tissue factor domain does not include lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind to factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments, the second multichain chimeric polypeptide does not stimulate coagulation in mammals. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide within the second multichain chimeric polypeptide further includes a signal sequence at its N-terminus. In some embodiments, the signal sequence includes SEQ ID NO: 28. In some embodiments, the signal sequence is SEQ ID NO: 28. In some embodiments, the affinity domain pair within the second multichain chimeric polypeptide is the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. In some embodiments, soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, soluble IL-15 includes a sequence that is 80% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 includes a sequence that is 90% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 includes a sequence that is 95% identical to SEQ ID NO: 26. In some embodiments, soluble IL-15 includes SEQ ID NO: 26. In some embodiments, the sushi domain of IL15Rα includes the sushi domain derived from human IL15Rα. In some embodiments, the sushi domain derived from human IL15Rα includes a sequence that is 80% identical to SEQ ID NO: 24.In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 90% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence that is 95% identical to SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. In some embodiments, the sushi domain derived from human IL15Rα is the mature full-length IL15Rα. In some embodiments, the affinity domain pairs within the second multi-chain chimeric polypeptide are selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first target-binding domain and the second target-binding domain within the second multi-chain chimeric polypeptide are each independently soluble IL-7 or soluble IL-21. In some embodiments, the first target-binding domain within the second multi-chain chimeric polypeptide specifically binds to the IL-21 receptor, and the second target-binding domain within the second multi-chain chimeric polypeptide specifically binds to the IL-7 receptor. In some embodiments, the first target-binding domain within the second multi-chain chimeric polypeptide specifically binds to the IL-7 receptor, and the second target-binding domain within the second multi-chain chimeric polypeptide specifically binds to the IL-21 receptor. In some embodiments, the first target-binding domain within the second multi-chain chimeric polypeptide comprises soluble IL-21. In some embodiments, the soluble IL-21 is soluble human IL-21. In some embodiments, the soluble human IL-21 comprises a sequence that is at least 80% identical to SEQ ID NO: 22. In some embodiments, the soluble human IL-21 comprises a sequence that is at least 90% identical to SEQ ID NO: 22. In some embodiments, the soluble human IL-21 comprises a sequence that is at least 95% identical to SEQ ID NO: 22. In some embodiments, the soluble human IL-21 comprises the sequence of SEQ ID NO: 22. In some embodiments, the second target-binding domain comprises soluble IL-7. 323. In some embodiments, the soluble IL-7 is soluble human IL-7.In some embodiments, soluble human IL-7 comprises a sequence that is at least 80% identical to SEQ ID NO: 23. In some embodiments, soluble human IL-7 comprises a sequence that is at least 90% identical to SEQ ID NO: 23. In some embodiments, soluble human IL-7 comprises a sequence that is at least 95% identical to SEQ ID NO: 23. In some embodiments, soluble human IL-7 comprises the sequence of SEQ ID NO: 23. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 68. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 70. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 71.In some embodiments, the first chimeric polypeptide within the second multi-chain chimeric polypeptide comprises SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide within the second multi-chain chimeric polypeptide comprises SEQ ID NO: 72. In some embodiments, the second chimeric polypeptide within the second multi-chain chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide within the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide within the second multi-chain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide within the second multi-chain chimeric polypeptide comprises SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide within the second multi-chain chimeric polypeptide comprises SEQ ID NO: 74. In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, and both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the linker domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, and one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the linker domain.

[0014] As used herein, the term "chimeric" refers to a polypeptide that contains amino acid sequences (e.g., domains) originally derived from two different sources (e.g., from the same or different species, e.g., two different naturally occurring proteins). For example, a chimeric polypeptide can contain domains from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can contain a domain that is a synthetic sequence (e.g., an scFv) and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide can contain at least two different domains that are synthetic sequences (e.g., two different scFvs).

[0015] The "multi-chain chimeric polypeptide" means a complex of two or more chimeric polypeptides.

[0016] The "antigen-binding domain" is one or more protein domains (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens. In some examples, the antigen-binding domain can bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen-binding domain can be an antibody or a fragment thereof. In some embodiments, the antigen-binding domain can include an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art.

[0017] The "soluble tissue factor domain" refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) with a segment of a wild-type mammalian tissue factor protein (e.g., wild-type human tissue factor protein) lacking a transmembrane domain and an intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

[0018] The term "soluble interleukin protein" is used herein to refer to a mature secreted interleukin protein or a biologically active fragment thereof. In some examples, the soluble interleukin protein can comprise a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a wild-type mature secreted mammalian interleukin protein (e.g., wild-type human interleukin protein) and retains its biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

[0019] The term "soluble cytokine protein" is used herein to refer to a mature secreted cytokine protein or a biologically active fragment thereof. In some examples, the soluble cytokine protein can comprise a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to a wild-type mature secreted mammalian interleukin protein (e.g., wild-type human interleukin protein) and retains its biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

[0020] The term "soluble interleukin receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding to 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 can be at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type interleukin receptor and retains the ability to specifically bind to one or more of its native ligands, and can include a sequence lacking its transmembrane domain (optionally further lacking an intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0021] The term "soluble cytokine receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding to one or more of its native ligands (e.g., under physiological conditions, such as in phosphate-buffered saline at room temperature). For example, a soluble cytokine receptor can be at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type cytokine receptor and retains the ability to specifically bind to one or more of its native ligands, and can include a sequence lacking its transmembrane domain (optionally further lacking an intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0022] The term "ligand of a costimulatory molecule" is used herein in its broadest sense to refer to a polypeptide that can bind to and activate a costimulatory receptor molecule on an immune cell (e.g., under physiological conditions, such as in phosphate-buffered saline at room temperature). For example, a ligand of a costimulatory molecule can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to a ligand of a costimulatory molecule that retains the ability to specifically bind to one or more of its native receptors. Non-limiting examples of ligands of costimulatory molecules are described herein.

[0023] The term "antibody" is used herein in its broadest sense and includes a particular type of immunoglobulin molecule that contains 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. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0024] "Affinity" refers to the total strength of non-covalent interactions between an antigen-binding site and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between a member of an antigen-binding domain and an antigen or epitope. The affinity of molecule X for its partner Y can be expressed by the dissociation equilibrium constant (KD). The kinetic components contributing to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including the methods described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity of an antigen-binding domain and its corresponding antigen or epitope are known in the art.

[0025] The term "affinity domain pair" refers to two different protein domains that specifically bind to each other with a K -7 less than 1×10 -8 M (e.g., less than 1×10 -9 M, less than 1×10 -10 M, less than 1×10 -11 M, or less than 1×10 D M). In some examples, an affinity domain pair can be a pair of naturally occurring proteins. In some embodiments, an affinity domain pair can be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs are described herein.

[0026] The term "epitope" means a part of an antigen that specifically binds to an antigen-binding domain. An epitope can consist of, for example, surface-accessible amino acid residues and / or sugar side chains, and can also have specific charge characteristics in addition to specific three-dimensional structural characteristics. Conformational epitopes and non-conformational epitopes are distinguished in that, in the presence of a denaturing solvent, the binding to the former may be lost, but the binding to the latter is unlikely to be lost. An epitope can include amino acid residues directly involved in the binding and other amino acid residues not directly involved in the binding. Methods for identifying the epitope to which an antigen-binding domain binds are known in the art.

[0027] The term "immune effector cell" refers to a cell of the mammalian immune system that can directly or indirectly recognize and / or cause the suppression of cell proliferation or cell death of pathogenic cells (e.g., cancer cells) in a mammal. Non-limiting examples of immune effector cells include macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes and T helper cells), neutrophils, monocytes, and eosinophils. Additional examples of immune effector cells are known in the art.

[0028] The term "treatment" means improving at least one symptom of a disorder. In some examples, the disorder being treated is cancer, and improving at least one symptom of cancer includes reducing the abnormal proliferation, gene expression, signal transduction, translation, and / or secretion of factors. Generally, the treatment method includes administering a therapeutically effective amount of a composition that reduces at least one symptom of the disorder to a subject in need of such treatment or determined to be in need of such treatment.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0030] [Invention 1001] A method for promoting the activation and proliferation of natural killer cells, comprising: (a) contacting natural killer cells with an effective amount of a first multichain chimeric polypeptide in a liquid culture medium for a first period under conditions that allow differentiation of the natural killer cells; (b) contacting the natural killer cells with an effective amount of (i) a second multichain chimeric polypeptide and (ii) an IgG1 antibody construct in a liquid culture medium for a second period under conditions that allow activation and proliferation of the natural killer cells; wherein: the first multichain chimeric polypeptide comprises a first chimeric polypeptide comprising a first target-binding domain, a soluble tissue factor domain, and a first domain of an affinity domain pair, and a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target-binding domain; the first chimeric polypeptide and the second chimeric polypeptide within the first multichain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the first multichain chimeric polypeptide, and the first target-binding domain and the second target-binding domain within the first multichain chimeric polypeptide each independently and specifically bind to an IL-18 receptor or an IL-12 receptor; the second multichain chimeric polypeptide comprises a first chimeric polypeptide comprising a first target-binding domain, a linker domain, and a first domain of an affinity domain pair, and a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target-binding domain; the first chimeric polypeptide and the second chimeric polypeptide within the second multichain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the second multichain chimeric polypeptide, and the first target-binding domain and the second target-binding domain within the second multichain chimeric polypeptide each independently and specifically bind to an IL-7 receptor or an IL-21 receptor, and the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to the linker domain; the method. [Invention 1002] ​ ​ The method of the present invention 1001, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide in the first multi-chain chimeric polypeptide. [The present invention 1003] The method of the present invention 1001, wherein the first chimeric polypeptide in the first multi-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide. [The present invention 1004] The method according to any one of the present inventions 1001 to 1003, wherein the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide in the first multi-chain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. [The present invention 1005] The method according to any one of the present inventions 1001 to 1003, wherein the first chimeric polypeptide in the first multi-chain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide. [The present invention 1006] The method according to any one of the present inventions 1001 to 1005, wherein the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide in the first multi-chain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. [The present invention 1007] The method according to any one of the present inventions 1001 to 1005, wherein the second chimeric polypeptide in the first multi-chain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide. [The present invention 1008] The method according to any one of the present inventions 1001 to 1007, wherein the soluble tissue factor domain is a soluble human tissue factor domain. [The present invention 1009] The method of the present invention 1008, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. [The present invention 1010] The method of the present invention 1009, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5. [The present invention 1011] The method of the present invention 1010, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. [The present invention 1012] The method of the present invention 1011, wherein the soluble human tissue factor domain contains SEQ ID NO: 5. [The present invention 1013] The method of the present invention 1008, wherein the soluble human tissue factor domain contains a sequence that is at least 80% identical to SEQ ID NO: 9. [The present invention 1014] The method of the present invention 1013, wherein the soluble human tissue factor domain contains a sequence that is at least 90% identical to SEQ ID NO: 9. [The present invention 1015] The method of the present invention 1014, wherein the soluble human tissue factor domain contains a sequence that is at least 95% identical to SEQ ID NO: 9. [The present invention 1016] The method of the present invention 1015, wherein the soluble human tissue factor domain contains SEQ ID NO: 9. [The present invention 1017] The method of the present invention 1008, wherein the soluble human tissue factor domain contains a sequence that is at least 80% identical to SEQ ID NO: 10. [The present invention 1018] The method of the present invention 1017, wherein the soluble human tissue factor domain contains a sequence that is at least 90% identical to SEQ ID NO: 10. [The present invention 1019] The method of the present invention 1018, wherein the soluble human tissue factor domain contains a sequence that is at least 95% identical to SEQ ID NO: 10. [The present invention 1020] The method of the present invention 1019, wherein the soluble human tissue factor domain contains SEQ ID NO: 10. [The present invention 1021] The soluble human tissue factor domain is lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein is not included in one or more of the methods of the present invention 1008 - 1011, 1013 - 1015, and 1017 - 1019. [The present invention 1022] The soluble human tissue factor domain is lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, a method according to the present invention 1021 that does not contain any of tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein A method according to the present invention 1021 that does not contain any of the above. [The present invention 1023] A method according to any one of the present inventions 1001 to 1022, wherein the soluble tissue factor domain cannot bind to factor VIIa. [The present invention 1024] A method according to any one of the present inventions 1001 to 1023, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa. [The present invention 1025] A method according to any one of the present inventions 1001 to 1024, wherein the first multi-chain chimeric polypeptide does not stimulate coagulation in mammals. [The present invention 1026] A method according to any one of the present inventions 1001 to 1025, wherein the first chimeric polypeptide and / or the second chimeric polypeptide within the first multi-chain chimeric polypeptide further comprises a signal sequence at its N-terminus. [The present invention 1027] A method according to the present invention 1026, wherein the signal sequence comprises SEQ ID NO: 28. [The present invention 1028] A method according to the present invention 1027, wherein the signal sequence is SEQ ID NO: 28. [The present invention 1029] A method according to any one of the present inventions 1001 to 1028, wherein the affinity domain pair within the first multi-chain chimeric polypeptide is the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. [The present invention 1030] A method according to the present invention 1029, wherein the soluble IL-15 has a D8N or D8A amino acid substitution. [The present invention 1031] A method according to the present invention 1029, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 26. [The present invention 1032] A method according to the present invention 1031, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 26. [The present invention 1033] A method according to the present invention 1032, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 26. [The present invention 1034] A method according to the present invention 1033, wherein the soluble IL-15 comprises SEQ ID NO: 26. [The present invention 1035] Any of the methods of the present invention from 1029 to 1034, wherein the sushi domain of IL15Rα comprises a sushi domain derived from human IL15Rα. [The present invention 1036] The method of the present invention 1035, wherein the sushi domain derived from human IL15Rα comprises a sequence that is 80% identical to SEQ ID NO: 24. [The present invention 1037] The method of the present invention 1036, wherein the sushi domain derived from human IL15Rα comprises a sequence that is 90% identical to SEQ ID NO: 24. [The present invention 1038] The method of the present invention 1037, wherein the sushi domain derived from human IL15Rα comprises a sequence that is 95% identical to SEQ ID NO: 24. [The present invention 1039] The method of the present invention 1038, wherein the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. [The present invention 1040] The method of the present invention 1035, wherein the sushi domain derived from human IL15Rα is mature full-length IL15Rα. [The present invention 1041] Any of the methods of the present invention from 1001 to 1028, wherein the affinity domain pair in the first multichain chimeric polypeptide is selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevin, and SNAP25. [The present invention 1042] Any of the methods of the present invention from 1001 to 1041, wherein the first target-binding domain and the second target-binding domain in the first multichain chimeric polypeptide are each independently soluble IL-15 or soluble IL-18. [The present invention 1043] Any of the methods of the present invention from 1001 to 1042, wherein the first target-binding domain in the first multichain chimeric polypeptide specifically binds to the IL-12 receptor, and the second target-binding domain in the first multichain chimeric polypeptide specifically binds to the IL-18 receptor. [The present invention 1044] Any of the methods of the present invention from 1001 to 1042, wherein the first target-binding domain in the first multichain chimeric polypeptide specifically binds to the IL-18 receptor, and the second target-binding domain in the first multichain chimeric polypeptide specifically binds to the IL-12 receptor. [The present invention 1045] The method of the present invention 1053, wherein the first target-binding domain in the first multichain chimeric polypeptide comprises soluble IL-18. [The present invention 1046] The method of the present invention 1045, wherein the soluble IL-18 is soluble human IL-18. [The present invention 1047] The method of the present invention 1046, wherein the soluble human IL-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 20. [The present invention 1048] The method of the present invention 1047, wherein the soluble human IL-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 20. [The present invention 1049] The method of the present invention 1048, wherein the soluble human IL-18 comprises a sequence that is at least 95% identical to SEQ ID NO: 20. [The present invention 1050] The method of the present invention 1049, wherein the soluble human IL-18 comprises the sequence of SEQ ID NO: 20. [The present invention 1051] The method according to any one of the present inventions 1044 to 1050, wherein the second target binding domain in the first multi-chain chimeric polypeptide comprises soluble IL-12. [The present invention 1052] The method of the present invention 1051, wherein the soluble IL-12 is soluble human IL-12. [The present invention 1053] The method of the present invention 1052, wherein the soluble human IL-12 comprises a soluble human IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence. [The present invention 1054] The method of the present invention 1053, wherein the soluble human IL-15 further comprises a linker sequence between the soluble IL-12β (p40) sequence and the soluble human IL-12α (p35) sequence. [The present invention 1055] The method of the present invention 1054, wherein the linker sequence comprises SEQ ID NO: 11. [The present invention 1056] The method according to any one of the present inventions 1053 to 1055, wherein the soluble human IL-12β (p40) sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 14. [The present invention 1057] The method of the present invention 1056, wherein the soluble human IL-12β (p40) sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 14. [The present invention 1058] The method of the present invention 1057, wherein the soluble human IL-12β (p40) sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 14. [The present invention 1059] The method of the present invention 1058, wherein the soluble human IL-12β (p40) sequence comprises SEQ ID NO: 14. [The present invention 1060] The method according to any one of the present inventions 1053 to 1059, wherein the soluble human IL-12α (p35) sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 16. [The present invention 1061] The method of the present invention 1060, wherein the soluble human IL-12α (p35) sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 16. [The present invention 1062] The method of the present invention 1061, wherein the soluble human IL-12α (p35) sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 16. [The present invention 1063] The method of the present invention 1062, wherein the soluble human IL-12α (p35) sequence comprises SEQ ID NO: 16. [The present invention 1064] The first chimeric polypeptide within the first multi-chain chimeric polypeptide TIFF0007713393000013.tif66138 The method of the present invention 1001, which comprises a sequence that is at least 80% identical to [The present invention 1065] The method of the present invention 1064, wherein the first chimeric polypeptide within the first multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 63. [The present invention 1066] The method of the present invention 1065, wherein the first chimeric polypeptide within the first multi-chain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 63. [The present invention 1067] The method of the present invention 1066, wherein the first chimeric polypeptide within the first multi-chain chimeric polypeptide comprises SEQ ID NO: 63. [The present invention 1068] The first chimeric polypeptide within the first multi-chain chimeric polypeptide TIFF0007713393000014.tif66138 The method of the present invention 1067, which comprises [The present invention 1069] The second chimeric polypeptide TIFF0007713393000015.tif79138 The method according to any one of the present inventions 1001 and 1064 to 1068, which comprises a sequence that is at least 80% identical to [The present invention 1070] The method of the present invention 1069, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 65. [The present invention 1071] The method of the present invention 1070, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 65. [The present invention 1072] The method of the present invention 1071, wherein the second chimeric polypeptide comprises SEQ ID NO: 65. [The present invention 1073] The second chimeric polypeptide TIFF0007713393000016.tif80138 The method of the present invention 1072, which comprises [The present invention 1074] The method according to any one of the present inventions 1001 to 1073, wherein the first target binding domain and the linker domain within the first chimeric polypeptide within the second multi-chain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. [The present invention 1075] The method according to any one of the present inventions 1001 to 1073, wherein the first chimeric polypeptide within the second multi-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain within the first chimeric polypeptide. [The present invention 1076] Any method according to any one of inventions 1001 to 1075, wherein the linker domain and the first domain of the pair of affinity domains are directly adjacent to each other within the first chimeric polypeptide in the second multi-chain chimeric polypeptide. [Invention 1077] Any method according to any one of inventions 1001 to 1075, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the first domain of the pair of affinity domains within the first chimeric polypeptide. [Invention 1078] Any method according to any one of inventions 1001 to 1077, wherein the second domain of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide in the second multi-chain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. [Invention 1079] Any method according to any one of inventions 1001 to 1077, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide. [Invention 1080] Any method according to any one of inventions 1001 to 1079, wherein the linker domain is a soluble tissue factor domain. [Invention 1081] The method of invention 1080, wherein the soluble tissue factor domain is a soluble human tissue factor domain. [Invention 1082] The method of invention 1081, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. [Invention 1083] The method of invention 1082, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5. [Invention 1084] The method of invention 1083, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. [Invention 1085] The method of invention 1084, wherein the soluble human tissue factor domain comprises SEQ ID NO: 5. [Invention 1086] The method of invention 1081, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 9. [Invention 1087] The method of invention 1086, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 9. [Invention 1088] The method of invention 1087, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. [The present invention 1089] The method of the present invention 1088, wherein the soluble human tissue factor domain comprises SEQ ID NO: 9. [The present invention 1090] The method of the present invention 1081, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10. [The present invention 1091] The method of the present invention 1090, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10. [The present invention 1092] The method of the present invention 1091, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 10. [The present invention 1093] The method of the present invention 1092, wherein the soluble human tissue factor domain comprises SEQ ID NO: 10. [The present invention 1094] The soluble human tissue factor domain is lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein and does not contain one or more of the above, and any of the methods of the present invention 1082-1084, 1086-1088, and 1090-1092. [The present invention 1095] The soluble human tissue factor domain is lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein The method of the present invention 1094 that does not include any of them. [The present invention 1096] Any method of the present inventions 1081 to 1095, wherein the soluble tissue factor domain cannot bind to factor VIIa. [The present invention 1097] Any method of the present inventions 1081 to 1096, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa. [The present invention 1098] Any method of the present inventions 1081 to 1097, wherein the second multichain chimeric polypeptide does not stimulate coagulation in mammals. [The present invention 1099] Any method of the present inventions 1001 to 1098, wherein the first chimeric polypeptide and / or the second chimeric polypeptide within the second multichain chimeric polypeptide further comprises a signal sequence at its N-terminus. [The present invention 1100] The method of the present invention 1099, wherein the signal sequence comprises SEQ ID NO: 28. [The present invention 1101] The method of the present invention 1100, wherein the signal sequence is SEQ ID NO: 28. [The present invention 1102] Any method of the present inventions 1001 to 1101, wherein the affinity domain pair within the second multichain chimeric polypeptide is the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. [The present invention 1103] The method of the present invention 1102, wherein the soluble IL-15 has a D8N or D8A amino acid substitution. [The present invention 1104] The method of the present invention 1102, wherein the soluble IL-15 comprises a sequence that is 80% identical to SEQ ID NO: 26. [The present invention 1105] The method of the present invention 1104, wherein the soluble IL-15 comprises a sequence that is 90% identical to SEQ ID NO: 26. [The present invention 1106] The method of the present invention 1105, wherein the soluble IL-15 comprises a sequence that is 95% identical to SEQ ID NO: 26. [The present invention 1107] The method of the present invention 1106, wherein the soluble IL-15 comprises SEQ ID NO: 26. [The present invention 1108] Any method of the present inventions 1102 to 1107, wherein the sushi domain of IL15Rα comprises the sushi domain derived from human IL15Rα. [The present invention 1109] The method of the present invention 1108, wherein the sushi domain derived from human IL15Rα comprises a sequence that is 80% identical to SEQ ID NO: 24. [The present invention 1110] The method of the present invention 1109, wherein the sushi domain derived from human IL15Rα comprises a sequence that is 90% identical to SEQ ID NO: 24. [The present invention 1111] The method of the present invention 1110, wherein the sushi domain derived from human IL15Rα comprises a sequence that is 95% identical to SEQ ID NO: 24. [The present invention 1112] The method of the present invention 1111, wherein the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. [The present invention 1113] The method of the present invention 1108, wherein the sushi domain derived from human IL15Rα is mature full-length IL15Rα. [The present invention 1114] The method according to any one of the present inventions 1001 to 1101, wherein the affinity domain pair in the second multichain chimeric polypeptide is selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevin, and SNAP25. [The present invention 1115] The method according to any one of the present inventions 1001 to 1114, wherein the first target-binding domain and the second target-binding domain in the second multichain chimeric polypeptide are each independently soluble IL-7 or soluble IL-21. [The present invention 1116] The method according to any one of the present inventions 1001 to 1115, wherein the first target-binding domain in the second multichain chimeric polypeptide specifically binds to the IL-21 receptor, and the second target-binding domain in the second multichain chimeric polypeptide specifically binds to the IL-7 receptor. [The present invention 1117] The method according to any one of the present inventions 1001 to 1115, wherein the first target-binding domain in the second multichain chimeric polypeptide specifically binds to the IL-7 receptor, and the second target-binding domain in the second multichain chimeric polypeptide specifically binds to the IL-21 receptor. [The present invention 1118] The method of the present invention 1117, wherein the first target-binding domain in the second multichain chimeric polypeptide comprises soluble IL-21. [The present invention 1119] The method of the present invention 1118, wherein the soluble IL-21 is soluble human IL-21. [The present invention 1120] The method of the present invention 1119, wherein the soluble human IL-21 comprises a sequence that is at least 80% identical to SEQ ID NO: 22. [The present invention 1121] The method of the present invention 1120, wherein the soluble human IL-21 comprises a sequence that is at least 90% identical to SEQ ID NO: 22. [The present invention 1122] The method of the present invention 1121, wherein the soluble human IL-21 comprises a sequence that is at least 95% identical to SEQ ID NO: 22. [The present invention 1123] The method of the present invention 1122, wherein the soluble human IL-21 comprises the sequence of SEQ ID NO: 22. [The present invention 1124] Any method of the present invention from 1117 to 1123, wherein the second target-binding domain comprises soluble IL-7. [The present invention 1125] The method of the present invention 1124, wherein the soluble IL-7 is soluble human IL-7. [The present invention 1126] The method of the present invention 1125, wherein the soluble human IL-7 comprises a sequence that is at least 80% identical to SEQ ID NO: 23. [The present invention 1127] The method of the present invention 1126, wherein the soluble human IL-7 comprises a sequence that is at least 90% identical to SEQ ID NO: 23. [The present invention 1128] The method of the present invention 1127, wherein the soluble human IL-7 comprises a sequence that is at least 95% identical to SEQ ID NO: 23. [The present invention 1129] The method of the present invention 1128, wherein the soluble human IL-7 comprises the sequence of SEQ ID NO: 23. [The present invention 1130] The first chimeric polypeptide within the second multichain chimeric polypeptide, TIFF0007713393000017.tif66138 comprises a sequence that is at least 80% identical to, the method of the present invention 1001. [The present invention 1131] The method of the present invention 1130, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 67. [The present invention 1132] The method of the present invention 1131, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 67. [The present invention 1133] The method of the present invention 1132, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 67. [The present invention 1134] The first chimeric polypeptide within the second multichain chimeric polypeptide, TIFF0007713393000018.tif66138 comprises, the method of the present invention 1133. [The present invention 1135] The second chimeric polypeptide within the second multichain chimeric polypeptide, TIFF0007713393000019.tif25138 comprises a sequence that is at least 80% identical to, any method of the present invention 1001 and 1130 to 1134. [The present invention 1136] The method of the present invention 1135, wherein the second chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 69. [The present invention 1137] The method of the present invention 1136, wherein the second chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 69. [The present invention 1138] The method of the present invention 1137, wherein the second chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 69. [The present invention 1139] The second chimeric polypeptide within the second multichain chimeric polypeptide, TIFF0007713393000020.tif31138 The method of the present invention 1138, comprising [The present invention 1140] wherein the first chimeric polypeptide within the second multichain chimeric polypeptide TIFF0007713393000021.tif66138 The method of the present invention 1001, comprising a sequence that is at least 80% identical to [The present invention 1141] The method of the present invention 1140, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 71. [The present invention 1142] The method of the present invention 1141, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 71. [The present invention 1143] The method of the present invention 1142, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 71. [The present invention 1144] wherein the first chimeric polypeptide within the second multichain chimeric polypeptide TIFF0007713393000022.tif66138 The method of the present invention 1143, comprising [The present invention 1145] wherein the second chimeric polypeptide within the second multichain chimeric polypeptide TIFF0007713393000023.tif32138 The method according to any one of the present invention 1001 and 1140 to 1144, comprising a sequence that is at least 80% identical to [The present invention 1146] The method of the present invention 1145, wherein the second chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 73. [The present invention 1147] The method of the present invention 1146, wherein the second chimeric polypeptide within the second multichain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 73. [The present invention 1148] The method of the present invention 1147, wherein the second chimeric polypeptide within the second multichain chimeric polypeptide comprises SEQ ID NO: 73. [The present invention 1149] wherein the second chimeric polypeptide within the second multichain chimeric polypeptide TIFF0007713393000024.tif31138 The method of the present invention 1148, comprising [The present invention 1150] The method according to any one of the present invention 1001 to 1149, wherein the IgG1 antibody construct is a monoclonal IgG1 antibody, and both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the linker domain. [The present invention 1151] The method according to any one of the present invention 1001 to 1149, wherein the IgG1 antibody construct is a bispecific IgG1 antibody, and one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the linker domain. [The present invention 1152] The method according to any one of the present invention 1001 to 1151, wherein the first period is from about 15 minutes to about 4 hours. [The present invention 1153] The method of the present invention 1152, wherein the first period is about 30 minutes to about 3.5 hours. [The present invention 1154] The method of the present invention 1153, wherein the first period is about 1 hour to about 3.5 hours. [The present invention 1155] The method of the present invention 1154, wherein the first period is about 2 hours to about 3.5 hours. [The present invention 1156] The method according to any one of the present inventions 1001 to 1155, wherein the liquid culture medium in step (a) and / or step (b) is a serum-containing liquid culture medium. [The present invention 1157] The NK cells are about 4.0×10 7 cells / mL to about 1.5×10 8 cells / mL and are present in the liquid culture medium in step (a). The method according to any one of the present inventions 1001 to 1156. [The present invention 1158] The NK cells are about 5.0×10 7 cells / mL to about 2.0×10 8 cells / mL and are present in the liquid culture medium in step (a). The method of the present invention 1157. [The present invention 1159] The method according to any one of the present inventions 1001 to 1158, wherein the second period is about 1 day to about 30 days. [The present invention 1160] The method of the present invention 1159, wherein the second period is about 7 days to about 30 days. [The present invention 1161] The method of the present invention 1160, wherein the second period is about 14 days to about 21 days. [The present invention 1162] The method according to any one of the present inventions 1001 to 1161, wherein the liquid culture medium in step (b) contains the second multi-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of about 0.5:1 to about 2:1. [The present invention 1163] The method of the present invention 1162, wherein the liquid culture medium in step (b) contains the second multi-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of about 0.8:1 to about 1.2:1. [The present invention 1164] The NK cells are present in the liquid culture medium in step (b) and are maintained at a density of about 0.5×10 6 cells / mL to about 2.0×10 6 cells / mL. The method according to any one of the present inventions 1001 to 1163. [The present invention 1165] The NK cells are present in the liquid culture medium in step (b) and are maintained at a density of about 1.0×10 6 cells / mL to about 2.0×10 6 cells / mL. The method of the present invention 1164. [The present invention 1166] The method according to any one of the present inventions 1001 to 1165, wherein the NK cells are those previously obtained from a subject. [The present invention 1167] The method according to any one of the present inventions 1001 to 1165, wherein the NK cells are those previously obtained from umbilical cord blood. [The present invention 1168] The method according to any one of inventions 1001 to 1165, wherein the NK cells are previously obtained from induced pluripotent stem cells. [Invention 1169] The method according to invention 1166 or 1167, further comprising obtaining the NK cells from the subject before step (a). [Invention 1170] The method according to any one of inventions 1001 to 1169, wherein the NK cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor. [Invention 1171] The method according to any one of inventions 1001 to 1169, further comprising introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells between step (a) and step (b). [Invention 1172] The method according to any one of inventions 1001 to 1169, further comprising introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells before step (a). [Invention 1173] The method according to any one of inventions 1001 to 1169, further comprising introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells during step (b). [Invention 1174] The method according to any one of inventions 1001 to 1173, further comprising isolating the NK cells after step (b). [Invention 1175] The method according to any one of inventions 1001 to 1174, wherein after step (b), the NK cells have an increased expression or secretion level of one or more proteins selected from the group consisting of TNF-α, IFN-γ, granzyme A, granzyme B, perforin, 2B4, CD8, CD11a, CD16, CD25, CD27, CD48, CD49d, CD54, CD56, CD58, CD62L, CD69, CD70, CD94, CD137, CD158a, CD158b, CD158e, CD178, CD226, CD253, NKG2C, NKG2D, LIR-1, LILR-B1, KIR2DL1, KIR3DL1, KIR2DL2, KIR2DL3, CXCR3, NKp30, NKp44, NKp46, NKG2D, DNAM-1, TRAIL, FasL, CXCR3, CXCR4, LTB, MX1, BAX, TNF-α, and IFN-γ as compared to the expression or secretion level of the one or more proteins before step (a). [Invention 1176] Any method of the present invention from 1001 to 1177, further comprising administering the NK cells to a subject in need thereof after the step of contacting. [The present invention 1177] The method of the present invention 1176, wherein the subject is identified or diagnosed as having an age-related disease or condition. [The present invention 1178] The method of the present invention 1177, wherein the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and kidney dysfunction. [The present invention 1179] The method of the present invention 1176, wherein the subject is identified or diagnosed as having cancer. [The present invention 1180] The method of the present invention 1179, wherein the cancer is selected from the group consisting of solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular cancer. [The present invention 1181] The method of the present invention 1176, wherein the subject is diagnosed or identified as having an infectious disease. [The present invention 1182] The method of the present invention 1181, wherein the infectious disease is an infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus. [The present invention 1183] Activated NK cells produced by any of the methods of the present invention 1001 to 1182. [The present invention 1184] A pharmaceutical composition comprising the activated NK cells of the present invention 1183. [The present invention 1185] A kit comprising a pharmaceutical composition comprising the activated NK cells of the present invention 1183. [The present invention 1186] A method of killing cancer cells, infected cells, or senescent cells in a subject in need of killing of cancer cells, infected cells, or senescent cells, the method comprising administering to the subject a therapeutically effective amount of the activated NK cells of the present invention 1183 or the pharmaceutical composition of the present invention 1184. [The present invention 1187] The method of the present invention 1186, wherein the subject is identified or diagnosed as having cancer. [The present invention 1188] The method of the present invention 1187, wherein the cancer is selected from the group consisting of solid tumors, hematological tumors, sarcomas, osteosarcomas, glioblastomas, neuroblastomas, melanomas, rhabdomyosarcomas, Ewing's sarcomas, osteosarcomas, B cell neoplasms, multiple myelomas, B cell lymphomas, B cell non-Hodgkin lymphomas, Hodgkin lymphomas, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndromes (MDS), cutaneous T cell lymphomas, retinoblastomas, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. [The present invention 1189] The method of the present invention 1186, wherein the subject is identified or diagnosed as having an age-related disease or condition. [The present invention 1190] The method of the present invention 1189, wherein the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, kidney transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and kidney dysfunction. [The present invention 1191] The method of the present invention 1186, wherein the subject is diagnosed or identified as having an infectious disease. [The present invention 1192] The method of the present invention 1191, wherein the infectious disease is infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus. [The present invention 1193] A method of treating a subject in need of treatment, the method comprising administering to the subject a therapeutically effective amount of the activated NK cells or activated T cells of the present invention 1183 or the pharmaceutical composition of the present invention 1184. [The present invention 1194] The method of the present invention 1193, wherein the subject is identified or diagnosed as having cancer. [The present invention 1195] The method of the present invention 1194, wherein the cancer is selected from the group consisting of solid tumor, hematological tumor, sarcoma, osteosarcoma, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B cell neoplasm, multiple myeloma, B cell lymphoma, B cell non-Hodgkin lymphoma, Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T cell lymphoma, retinoblastoma, gastric cancer, urothelial cancer, lung cancer, renal cell cancer, gastroesophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, squamous cell head and neck cancer, endometrial cancer, cervical cancer, liver cancer, and hepatocellular cancer. [The present invention 1196] The method of the present invention 1193, wherein the subject is identified or diagnosed as having an age-related disease or condition. [The present invention 1197] The method of the present invention 1196, wherein the age-related disease or condition is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipoatrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. [The present invention 1198] The method of the present invention 1197, wherein the subject is diagnosed or identified as having an infectious disease. [The present invention 1199] The method of the present invention 1198, wherein the infectious disease is an infection by human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, and hepatitis C virus, papillomavirus, and influenza virus. [The present invention 1200] (a) A first multichain chimeric polypeptide, A first chimeric polypeptide comprising a first target-binding domain, a soluble tissue factor domain, and a first domain of an affinity domain pair, and A second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target-binding domain wherein the first chimeric polypeptide and the second chimeric polypeptide within the first multichain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the first multichain chimeric polypeptide, and the first target-binding domain and the second target-binding domain within the first multichain chimeric polypeptide each independently and specifically bind to the IL-18 receptor or the IL-12 receptor, the first multichain chimeric polypeptide, and (b) A second multichain chimeric polypeptide, A first chimeric polypeptide comprising a first target binding domain, a linker domain, and a first domain of an affinity domain pair, and a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target binding domain comprising the first chimeric polypeptide and the second chimeric polypeptide within the second multichain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the second multichain chimeric polypeptide, and the first target binding domain and the second target binding domain within the second multichain chimeric polypeptide each independently and specifically bind to an IL-7 receptor or an IL-21 receptor, the second multichain chimeric polypeptide, and (c) an IgG1 antibody comprising at least one antigen binding domain that specifically binds to the linker domain A kit comprising [Invention 1201] The kit according to Invention 1200, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide in the first multichain chimeric polypeptide. [Invention 1202] The kit according to Invention 1200, wherein the first chimeric polypeptide in the first multichain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide. [Invention 1203] The kit according to any one of Inventions 1200 to 1202, wherein the soluble tissue factor domain and the first domain of the affinity domain pair within the first chimeric polypeptide in the first multichain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. [Invention 1204] The kit according to any one of Inventions 1200 to 1202, wherein the first chimeric polypeptide in the first multichain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide. [Invention 1205] The kit according to any one of Inventions 1200 to 1204, wherein the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide in the first multichain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. [Invention 1206] The kit according to any one of 1200 to 1204 of the present invention, wherein the second chimeric polypeptide in the first multi-chain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair in the second chimeric polypeptide and the second target binding domain. [The present invention 1207] The kit according to any one of 1200 to 1206 of the present invention, wherein the soluble tissue factor domain is a soluble human tissue factor domain. [The present invention 1208] The kit according to 1207 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. [The present invention 1209] The kit according to 1208 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5. [The present invention 1210] The kit according to 1209 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. [The present invention 1211] The kit according to 1210 of the present invention, wherein the soluble human tissue factor domain comprises SEQ ID NO: 5. [The present invention 1212] The kit according to 1207 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 9. [The present invention 1213] The kit according to 1212 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 9. [The present invention 1214] The kit according to 1213 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. [The present invention 1215] The kit according to 1214 of the present invention, wherein the soluble human tissue factor domain comprises SEQ ID NO: 9. [The present invention 1216] The kit according to 1207 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10. [The present invention 1217] The kit according to 1216 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10. [The present invention 1218] The kit according to 1217 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 10. [The present invention 1219] The kit according to 1218 of the present invention, wherein the soluble human tissue factor domain comprises SEQ ID NO: 10. [The present invention 1220] The soluble human tissue factor domain is lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, Tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein One or more of which are not included, any kit of the present invention 1207-1210, 1212-1214, and 1216-1218. [The present invention 1221] Wherein the soluble human tissue factor domain is lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein None of which are included, the kit of the present invention 1220. [The present invention 1222] Any kit of the present invention 1200-1221, wherein the soluble tissue factor domain cannot bind to factor VIIa. [The present invention 1223] Any kit of the present invention 1200-1222, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa. [The present invention 1224] Any kit of the present invention 1200-1223, wherein the first multichain chimeric polypeptide does not stimulate coagulation in mammals. [The present invention 1225] Any kit of the present invention 1200-1224, wherein the first chimeric polypeptide and / or the second chimeric polypeptide within the first multichain chimeric polypeptide further comprises a signal sequence at its N-terminus. [The present invention 1226] The kit of the present invention 1225, wherein the signal sequence comprises SEQ ID NO: 28. [The present invention 1227] The kit of the present invention 1226, wherein the signal sequence is SEQ ID NO: 28. [The present invention 1228] The kit of any one of the present inventions 1200 to 1227, wherein the pair of affinity domains in the first multi-chain chimeric polypeptide is the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. [The present invention 1229] The kit of the present invention 1228, wherein the soluble IL-15 has a D8N or D8A amino acid substitution. [The present invention 1230] The kit of the present invention 1228, wherein the soluble IL-15 comprises a sequence 80% identical to SEQ ID NO: 26. [The present invention 1231] The kit of the present invention 1230, wherein the soluble IL-15 comprises a sequence 90% identical to SEQ ID NO: 26. [The present invention 1232] The kit of the present invention 1231, wherein the soluble IL-15 comprises a sequence 95% identical to SEQ ID NO: 26. [The present invention 1233] The kit of the present invention 1232, wherein the soluble IL-15 comprises SEQ ID NO: 26. [The present invention 1234] The kit of any one of the present inventions 1228 to 1233, wherein the sushi domain of IL15Rα comprises the sushi domain derived from human IL15Rα. [The present invention 1235] The kit of the present invention 1234, wherein the sushi domain derived from human IL15Rα comprises a sequence 80% identical to SEQ ID NO: 24. [The present invention 1236] The kit of the present invention 1235, wherein the sushi domain derived from human IL15Rα comprises a sequence 90% identical to SEQ ID NO: 24. [The present invention 1237] The kit of the present invention 1236, wherein the sushi domain derived from human IL15Rα comprises a sequence 95% identical to SEQ ID NO: 24. [The present invention 1238] The kit of the present invention 1237, wherein the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. [The present invention 1239] The kit of the present invention 1234, wherein the sushi domain derived from human IL15Rα is the mature full-length IL15Rα. [The present invention 1240] The kit of any one of the present inventions 1200 to 1227, wherein the pair of affinity domains in the first multi-chain chimeric polypeptide is selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. [The present invention 1241] The kit according to any one of the inventions 1200 to 1240, wherein the first target binding domain and the second target binding domain in the first multi-chain chimeric polypeptide are each independently soluble IL-15 or soluble IL-18. [Invention 1242] The kit according to any one of the inventions 1200 to 1241, wherein the first target binding domain in the first multi-chain chimeric polypeptide specifically binds to the IL-12 receptor, and the second target binding domain in the first multi-chain chimeric polypeptide specifically binds to the IL-18 receptor. [Invention 1243] The kit according to any one of the inventions 1200 to 1241, wherein the first target binding domain in the first multi-chain chimeric polypeptide specifically binds to the IL-18 receptor, and the second target binding domain in the first multi-chain chimeric polypeptide specifically binds to the IL-12 receptor. [Invention 1244] The kit according to invention 1252, wherein the first target binding domain in the first multi-chain chimeric polypeptide comprises soluble IL-18. [Invention 1245] The kit according to invention 1244, wherein the soluble IL-18 is soluble human IL-18. [Invention 1246] The kit according to invention 1245, wherein the soluble human IL-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 20. [Invention 1247] The kit according to invention 1246, wherein the soluble human IL-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 20. [Invention 1248] The kit according to invention 1247, wherein the soluble human IL-18 comprises a sequence that is at least 95% identical to SEQ ID NO: 20. [Invention 1249] The kit according to invention 1248, wherein the soluble human IL-18 comprises the sequence of SEQ ID NO: 20. [Invention 1250] The kit according to any one of the inventions 1243 to 1249, wherein the second target binding domain in the first multi-chain chimeric polypeptide comprises soluble IL-12. [Invention 1251] The kit according to invention 1250, wherein the soluble IL-12 is soluble human IL-12. [Invention 1252] The kit according to invention 1251, wherein the soluble human IL-12 comprises a soluble human IL-12β (p40) sequence and a soluble human IL-12α (p35) sequence. [Invention 1253] The kit according to invention 1252, wherein the soluble human IL-15 further comprises a linker sequence between the soluble IL-12β (p40) sequence and the soluble human IL-12α (p35) sequence. [Invention 1254] The kit of the present invention 1253, wherein the linker array contains SEQ ID NO: 11. [The present invention 1255] The kit of any one of the present inventions 1252 to 1254, wherein the soluble human IL-12β (p40) sequence contains a sequence that is at least 80% identical to SEQ ID NO: 14. [The present invention 1256] The kit of the present invention 1255, wherein the soluble human IL-12β (p40) sequence contains a sequence that is at least 90% identical to SEQ ID NO: 14. [The present invention 1257] The kit of the present invention 1256, wherein the soluble human IL-12β (p40) sequence contains a sequence that is at least 95% identical to SEQ ID NO: 14. [The present invention 1258] The kit of the present invention 1257, wherein the soluble human IL-12β (p40) sequence contains SEQ ID NO: 14. [The present invention 1259] The kit of any one of the present inventions 1252 to 1258, wherein the soluble human IL-12α (p35) sequence contains a sequence that is at least 80% identical to SEQ ID NO: 16. [The present invention 1260] The kit of the present invention 1259, wherein the soluble human IL-12α (p35) sequence contains a sequence that is at least 90% identical to SEQ ID NO: 16. [The present invention 1261] The kit of the present invention 1260, wherein the soluble human IL-12α (p35) sequence contains a sequence that is at least 95% identical to SEQ ID NO: 16. [The present invention 1262] The kit of the present invention 1261, wherein the soluble human IL-12α (p35) sequence contains SEQ ID NO: 16. [The present invention 1263] The kit of the present invention 1200, wherein the first chimeric polypeptide in the first multichain chimeric polypeptide contains a sequence that is at least 80% identical to SEQ ID NO: 63. [The present invention 1264] The kit of the present invention 1263, wherein the first chimeric polypeptide in the first multichain chimeric polypeptide contains a sequence that is at least 90% identical to SEQ ID NO: 63. [The present invention 1265] The kit of the present invention 1264, wherein the first chimeric polypeptide in the first multichain chimeric polypeptide contains a sequence that is at least 95% identical to SEQ ID NO: 63. [The present invention 1266] The kit of the present invention 1265, wherein the first chimeric polypeptide in the first multichain chimeric polypeptide contains SEQ ID NO: 63. [The present invention 1267] The kit of the present invention 1266, wherein the first chimeric polypeptide in the first multichain chimeric polypeptide contains SEQ ID NO: 64. [The present invention 1268] The kit of any one of the present inventions 1200 and 1263 to 1267, wherein the second chimeric polypeptide contains a sequence that is at least 80% identical to SEQ ID NO: 65. [The present invention 1269] The kit of the present invention 1268, wherein the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 65. [The present invention 1270] The kit of the present invention 1269, wherein the second chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 65. [The present invention 1271] The kit of the present invention 1270, wherein the second chimeric polypeptide comprises SEQ ID NO: 65. [The present invention 1272] The kit of the present invention 1271, wherein the second chimeric polypeptide comprises SEQ ID NO: 66. [The present invention 1273] The kit according to any one of the present inventions 1200 to 1272, wherein the first target binding domain and the linker domain in the first chimeric polypeptide within the second multi-chain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. [The present invention 1274] The kit according to any one of the present inventions 1200 to 1272, wherein the first chimeric polypeptide within the second multi-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the linker domain in the first chimeric polypeptide. [The present invention 1275] The kit according to any one of the present inventions 1200 to 1274, wherein the linker domain and the first domain of the affinity domain pair are directly adjacent to each other within the first chimeric polypeptide within the second multi-chain chimeric polypeptide. [The present invention 1276] The kit according to any one of the present inventions 1200 to 1274, wherein the first chimeric polypeptide within the second multi-chain chimeric polypeptide further comprises a linker sequence between the linker domain and the first domain of the affinity domain pair in the first chimeric polypeptide. [The present invention 1277] The kit according to any one of the present inventions 1200 to 1276, wherein the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide within the second multi-chain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide. [The present invention 1278] The kit according to any one of the present inventions 1200 to 1276, wherein the second chimeric polypeptide within the second multi-chain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide. [The present invention 1279] The kit according to any one of 1200 to 1278 of the present invention, wherein the linker domain is a soluble tissue factor domain. [The present invention 1280] The kit according to 1279 of the present invention, wherein the soluble tissue factor domain is a soluble human tissue factor domain. [The present invention 1281] The kit according to 1280 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 5. [The present invention 1282] The kit according to 1281 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 5. [The present invention 1283] The kit according to 1282 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 5. [The present invention 1284] The kit according to 1283 of the present invention, wherein the soluble human tissue factor domain comprises SEQ ID NO: 5. [The present invention 1285] The kit according to 1280 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 9. [The present invention 1286] The kit according to 1285 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 9. [The present invention 1287] The kit according to 1284 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 9. [The present invention 1288] The kit according to 1285 of the present invention, wherein the soluble human tissue factor domain comprises SEQ ID NO: 9. [The present invention 1289] The kit according to 1278 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10. [The present invention 1290] The kit according to 1289 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10. [The present invention 1291] The kit according to 1290 of the present invention, wherein the soluble human tissue factor domain comprises a sequence that is at least 95% identical to SEQ ID NO: 3. [The present invention 1292] The kit according to 1291 of the present invention, wherein the soluble human tissue factor domain comprises SEQ ID NO: 10. [The present invention 1293] The soluble human tissue factor domain is lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, Tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein A kit according to any one of aspects 1279-1281, 1283-1285, and 1289-1291 of the present invention, which does not contain one or more of the above. [Aspect 1294 of the present invention] Wherein the soluble human tissue factor domain has lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, has isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, has tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, has aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, has tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, has arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and has phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein A kit according to aspect 1293 of the present invention, which does not contain any of the above. [Aspect 1295 of the present invention] A kit according to any one of aspects 1278-1294 of the present invention, wherein the soluble tissue factor domain cannot bind to factor VIIa. [Aspect 1296 of the present invention] A kit according to any one of aspects 1278-1295 of the present invention, wherein the soluble tissue factor domain does not convert inactive factor X to factor Xa. [Aspect 1297 of the present invention] A kit according to any one of aspects 1278-1296 of the present invention, wherein the second multichain chimeric polypeptide does not stimulate coagulation in mammals. [Aspect 1298 of the present invention] A kit according to any one of aspects 1198-1297 of the present invention, wherein the first chimeric polypeptide and / or the second chimeric polypeptide within the second multichain chimeric polypeptide further comprises a signal sequence at its N-terminus. [Aspect 1299 of the present invention] A kit according to aspect 1298 of the present invention, wherein the signal sequence comprises SEQ ID NO: 28. [Aspect 1300 of the present invention] A kit according to aspect 1299 of the present invention, wherein the signal sequence is SEQ ID NO: 28. [Aspect 1301 of the present invention] The kit according to any one of inventions 1198 to 1298 of the present invention, wherein the pair of affinity domains in the second multi-chain chimeric polypeptide is a sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. [Invention 1302] The kit according to invention 1301 of the present invention, wherein the soluble IL-15 has a D8N or D8A amino acid substitution. [Invention 1303] The kit according to invention 1301 of the present invention, wherein the soluble IL-15 comprises a sequence 80% identical to SEQ ID NO: 26. [Invention 1304] The kit according to invention 1303 of the present invention, wherein the soluble IL-15 comprises a sequence 90% identical to SEQ ID NO: 26. [Invention 1305] The kit according to invention 1304 of the present invention, wherein the soluble IL-15 comprises a sequence 95% identical to SEQ ID NO: 26. [Invention 1306] The kit according to invention 1305 of the present invention, wherein the soluble IL-15 comprises SEQ ID NO: 26. [Invention 1307] The kit according to any one of inventions 1301 to 1306 of the present invention, wherein the sushi domain of IL15Rα comprises a sushi domain derived from human IL15Rα. [Invention 1308] The kit according to invention 1307 of the present invention, wherein the sushi domain derived from human IL15Rα comprises a sequence 80% identical to SEQ ID NO: 24. [Invention 1309] The kit according to invention 1308 of the present invention, wherein the sushi domain derived from human IL15Rα comprises a sequence 90% identical to SEQ ID NO: 24. [Invention 1310] The kit according to invention 1309 of the present invention, wherein the sushi domain derived from human IL15Rα comprises a sequence 95% identical to SEQ ID NO: 24. [Invention 1311] The kit according to invention 1310 of the present invention, wherein the sushi domain derived from human IL15Rα comprises SEQ ID NO: 24. [Invention 1312] The kit according to invention 1307 of the present invention, wherein the sushi domain derived from human IL15Rα is mature full-length IL15Rα. [Invention 1313] The kit according to any one of inventions 1200 to 1300 of the present invention, wherein the pair of affinity domains in the second multi-chain chimeric polypeptide is selected from the group consisting of barnase and barstar, PKA and AKAP, an adapter / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevin, and SNAP25. [Invention 1314] The kit according to any one of inventions 1200 to 1313 of the present invention, wherein the first target-binding domain and the second target-binding domain in the second multi-chain chimeric polypeptide are each independently soluble IL-7 or soluble IL-21. [The present invention 1315] The kit according to any one of inventions 1200 to 1314, wherein the first target binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-21 receptor, and the second target binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-7 receptor. [The present invention 1316] The kit according to any one of inventions 1200 to 1314, wherein the first target binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-7 receptor, and the second target binding domain in the second multi-chain chimeric polypeptide specifically binds to the IL-21 receptor. [The present invention 1317] The kit according to invention 1316, wherein the first target binding domain in the second multi-chain chimeric polypeptide contains soluble IL-21. [The present invention 1318] The kit according to invention 1317, wherein the soluble IL-21 is soluble human IL-21. [The present invention 1319] The kit according to invention 1318, wherein the soluble human IL-21 contains a sequence that is at least 80% identical to SEQ ID NO: 22. [The present invention 1320] The kit according to invention 1319, wherein the soluble human IL-21 contains a sequence that is at least 90% identical to SEQ ID NO: 22. [The present invention 1321] The kit according to invention 1320, wherein the soluble human IL-21 contains a sequence that is at least 95% identical to SEQ ID NO: 22. [The present invention 1322] The kit according to invention 1321, wherein the soluble human IL-21 contains the sequence of SEQ ID NO: 22. [The present invention 1323] The kit according to any one of inventions 1316 to 1322, wherein the second target binding domain contains soluble IL-7. [The present invention 1324] The kit according to invention 1323, wherein the soluble IL-7 is soluble human IL-7. [The present invention 1325] The kit according to invention 1324, wherein the soluble human IL-7 contains a sequence that is at least 80% identical to SEQ ID NO: 23. [The present invention 1326] The kit according to invention 1325, wherein the soluble human IL-7 contains a sequence that is at least 90% identical to SEQ ID NO: 23. [The present invention 1327] The kit according to invention 1326, wherein the soluble human IL-7 contains a sequence that is at least 95% identical to SEQ ID NO: 23. [The present invention 1328] The kit according to invention 1327, wherein the soluble human IL-7 contains the sequence of SEQ ID NO: 23. [The present invention 1329] The kit according to invention 1200, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide contains a sequence that is at least 80% identical to SEQ ID NO: 67. [The present invention 1330] The kit of the invention 1329, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 67. [Invention 1331] The kit of the invention 1330, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 67. [Invention 1332] The kit of the invention 1331, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 67. [Invention 1333] The kit of the invention 1332, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 68. [Invention 1334] The kit of any one of the inventions 1200 and 1329 - 1333, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 69. [Invention 1335] The kit of the invention 1334, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 69. [Invention 1336] The kit of the invention 1335, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 69. [Invention 1337] The kit of the invention 1336, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 69. [Invention 1338] The kit of the invention 1337, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 70. [Invention 1339] The kit of the invention 1200, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 71. [Invention 1340] The kit of the invention 1339, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 71. [Invention 1341] The kit of the invention 1340, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence that is at least 95% identical to SEQ ID NO: 71. [Invention 1342] The kit of the invention 1341, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 71. [Invention 1343] The kit of the present invention 1342, wherein the first chimeric polypeptide in the second multi-chain chimeric polypeptide contains SEQ ID NO: 72. [The present invention 1344] The kit of any one of the present inventions 1200 and 1339 - 1343, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide contains a sequence that is at least 80% identical to SEQ ID NO: 73. [The present invention 1345] The kit of the present invention 1344, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide contains a sequence that is at least 90% identical to SEQ ID NO: 73. [The present invention 1346] The kit of the present invention 1345, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide contains a sequence that is at least 95% identical to SEQ ID NO: 73. [The present invention 1347] The kit of the present invention 1346, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide contains SEQ ID NO: 73. [The present invention 1348] The kit of the present invention 1347, wherein the second chimeric polypeptide in the second multi-chain chimeric polypeptide contains SEQ ID NO: 74. [The present invention 1349] The kit of any one of the present inventions 1200 - 1348, wherein the IgG1 antibody construct is a monoclonal IgG1 antibody, and both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the linker domain. [The present invention 1350] The kit of any one of the present inventions 1200 - 1348, wherein the IgG1 antibody construct is a bispecific IgG1 antibody, and one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the linker domain. Other features and advantages of the present invention will become apparent from the following detailed description of the invention, the drawings, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0031]

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Mode for Carrying Out the Invention

[0032] Detailed Description Specific combinations of two multi-chain chimeric polypeptides, their use, and kits containing the same are provided herein. The multi-chain chimeric polypeptide includes a linker domain disposed between two target binding domains. In some embodiments, the linker domain is or includes a soluble tissue factor domain. In some embodiments, the linker domain can be recognized by a cognate IgG1 antibody (e.g., a monoclonal antibody). In some embodiments, a combination of two of the multi-chain chimeric polypeptides described herein can be used to stimulate NK cells, induce or increase the proliferation of NK cells, and / or induce the differentiation of NK cells, and thereafter, the NK cells can be used to treat a subject.

[0033] Methods for promoting the activation and proliferation of natural killer cells are provided herein. The methods include: (a) contacting natural killer cells with an effective amount of a first multi-chain chimeric polypeptide in a liquid culture medium (e.g., any of the liquid culture media described herein) that allows for the differentiation of natural killer cells, for a first period of time, under conditions that allow for the differentiation of natural killer cells; and (b) contacting natural killer cells with an effective amount of (i) a second multi-chain chimeric polypeptide and (ii) an IgG1 antibody construct (e.g., any of the exemplary IgG1 antibody constructs described herein) that includes at least one antigen-binding domain that specifically binds to a linker domain, in a liquid culture medium, for a second period of time, under conditions that allow for the activation and proliferation of NK cells. Also provided are NK cells produced by any of these methods, and methods of treating a subject using these NK cells. Compositions comprising any of the NK cells generated using these methods are also provided herein. Also provided is a kit comprising: (a) a first multi-chain chimeric polypeptide (e.g., any of the exemplary first multi-chain chimeric polypeptides described herein) that includes a first and a second chimeric polypeptide; (b) a second multi-chain chimeric polypeptide (e.g., any of the exemplary second multi-chain chimeric polypeptides described herein) that includes a first and a second chimeric polypeptide; and (c) an IgG1 antibody construct (e.g., any of the IgG1 antibody constructs described herein) that includes at least one antigen-binding domain that specifically binds to a linker domain. Exemplary embodiments of the methods provided herein are shown in FIGS. 30-32.

[0034] Current ex vivo NK expansion methods can include feeder cell-based expansion methods and cytokine-based expansion methods. Exemplary disadvantages of feeder cell-based expansion methods include the development and maintenance of GMP-grade feeder cells and methods demonstrating effective feeder cell irradiation, the need to deplete all feeder cell contaminants containing DNA from the final product prior to administration to a patient, and the long and costly manufacturing process. Exemplary disadvantages of cytokine-based expansion methods include sub-optimal purity of the final NK cell product, fewer final NK cell numbers compared to feeder cell expansion, and the cost of recombinant cytokines and the lack of commercially available GMP-grade cytokines other than IL-2. The methods described herein provide advantages such as the generation of highly activated NK cells from a patient, large-scale expansion comparable to or similar to the feeder cell method, cost-effective expansion and activation with limited processing requirements, and the ability to inject the cells into a subject, thereby enabling in vivo cell expansion.

[0035] In some embodiments, the methods described herein can induce the generation and expansion of NK cells that are highly activated with long persistence and high cytotoxicity against diseased cells in a recipient host (e.g., a cell having any of the exemplary aspects of the memory-like natural killer cells described herein). In some embodiments of any of the methods described herein, the contacting step comprising contacting the NK cells with a first multi-chain chimeric polypeptide results in less than 50% cell death (e.g., apoptosis) (e.g., less than 48% cell death, less than 46% cell death, less than 44% cell death, less than 42% cell death, less than 40% cell death, less than 38% cell death, less than 36% cell death, less than 34% cell death, less than 32% cell death, less than 30% cell death, less than 28% cell death, less than 26% cell death, less than 24% cell death, less than 22% cell death, less than 20% cell death, less than 18% cell death, less than 16% cell death, less than 14% cell death, less than 12% cell death, less than 10% cell death, less than 8% cell death, less than 6% cell death, less than 4% cell death, or less than 2% cell death).

[0036] In some embodiments, by any of the methods described herein, prior to performing any of the methods described herein (e.g., before contacting NK cells with any of the exemplary first multi-chain chimeric polypeptides described herein), compared to NK cells, there is an increase in one or more of glycolysis, aerobic metabolism, oxidative phosphorylation, glucose consumption, and extracellular acidification rate (ECAR) (e.g., an increase of about 1% to about 5,000%, about 1% to about 4,000%, about 1% to about 3,000%, about 1% to about 2,000%, about 1% to about 1,000%, about 1% to about 800%, about 1% to about 600%, about 1% to about 500%, about 1% to about 400%, about 1% to about 300%, 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 80%, about 1% to about 60%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 1% to about 5%, about 5% to about 5,000%, about 5% to about 4,000%, about 5% to about 3,000%, about 5% to about 2,000%, about 5% to about 1,000%, about 5% to about 800%, about 5% to about 600%, about 5% to about 500%, about 5% to about 400%, about 5% to about 300%, 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 80%, about 5% to about 60%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 5,000%, about 10% to about 4,000%, about 10% to about 3,000%, about 10% to about 2,000%, about 10% to about 1,000% increase, about 10% to about 800% increase, about 10% to about 600% increase, about 10% to about 500% increase, about 10% to about 400% increase, about 10% to about 300% increase, about 10% to about 200% increase, about 10% to about 180% increase, about 10% to about 160% increase, about 10% to about 140% increase, about 10% to about 120% increase, about 10% to about 100% increase, about 10% to about 80% increase, about 10% to about 60% increase, about 10% to about 40% increase, about 10% to about 30% increase, about 10% to about 20% increase, about 10% to about 15% increase, about 15% to about 5,000% increase, about 15% to about 4,000% increase, about 15% to about 3,000% increase, about 15% to about 2,000% increase, about 15% to about 1,000% increase, about 15% to about 800% increase, about 15% to about 600% increase, about 15% to about 500% increase, about 15% to about 400% increase, about 15% to about 300% increase, about 15% to about 200% increase, about 15% to about 180% increase, about 15% to about 160% increase, about 15% to about 140% increase, about 15% to about 120% increase, about 15% to about 100% increase, about 15% to about 80% increase, about 15% to about 60% increase, about 15% to about 40% increase, about 15% to about 30% increase, about 15% to about 20% increase, about 20% to about 5,000% increase, about 20% to about 4,000% increase, about 20% to about 3,000% increase, about 20% to about 2,000% increase, about 20% to about 1,000% increase, about 20% to about 800% increase, about 20% to about 600% increase, about 20% to about 500% increase, about 20% to about 400% increase, about 20% to about 300% increase, about 20% to about 200% increase, about 20% to about 180% increase, about 20% to about 160% increase, about 20% to about 140% increase, about 20% to about 120% increase, about 20% to about 100% increase, about 20% to about 80% increase, about 20% to about 60% increase, about 20% to about 40% increase, about 20% to about 30% increase, about 30% to about 5,000% increase, about 30% to about 4,000% increase, about 30% to about 3,000% increase, about 30% to about 2,000% increase, about 30% to about 1,000% increase, about 30% to about 800% increase, about 30% to about 600% increase, about 30% to about 500% increase, about 30% to about 400% increase, about 30% to about 300% increase, about 30% to about 200% increase, about 30% to about 180% increase, about 30% to about 160% increase, about 30% to about 140% increase, about 30% to about 120% increase, about 30% to about 100% increase, about 30% to about 80% increase, about 30% to about 60% increase, about 30% to about 40% increase, about 40% to about 5,000% increase, about 40% to about 4,000% increase, about 40% to about 3,000% increase, about 40% to about 2,000% increase, about 40% to about 1,000% increase, about 40% to about 800% increase, about 40% to about 600% increase, about 40% to about 500% increase, about 40% to about 400% increase, about 40% to about 300% increase, about 40% to about 200% increase, about 40% to about 180% increase, about 40% to about 160% increase, about 40% to about 140% increase, about 40% to about 120% increase, about 40% to about 100% increase, about 40% to about 80% increase, about 40% to about 60% increase, about 60% to about 5,000% increase, about 60% to about 4,000% increase, about 60% to about 3,000% increase, about 60% to about 2,000% increase, about 60% to about 1,000% increase, about 60% to about 800% increase, about 60% to about 600% increase, about 60% to about 500% increase, about 60% to about 400% increase, about 60% to about 300% increase, about 60% to about 200% increase, about 60% to about 180% increase, about 60% to about 160% increase, about 60% to about 140% increase, about 60% to about 120% increase, about 60% to about 100% increase, about 60% to about 80% increase, about 80% to about 5,000% increase, about 80% to about 4,000% increase, about 80% to about 3,000% increase, about 80% to about 2,000% increase, about 80% to about 1,000% increase, about 80% to about 800% increase, about 80% to about 600% increase, about 80% to about 500% increase, about 80% to about 400% increase, about 80% to about 300% increase, about 80% to about 200% increase, about 80% to about 180% increase, about 80% to about 160% increase, about 80% to about 140% increase, about 80% to about 120% increase, about 80% to about 100% increase, about 100% to about 5,000% increase, about 100% to about 4,000% increase, about 100% to about 3,000% increase, about 100% to about 2,000% increase, about 100% to about 1,000% increase, about 100% - about 800% increase, about 100% - about 600% increase, about 100% - about 500% increase, about 100% - about 400% increase, about 100% - about 300% increase, about 100% - about 200% increase, about 100% - about 180% increase, about 100% - about 160% increase, about 100% - about 140% increase, about 100% - about 120% increase, about 120% - about 5,000% increase, about 120% - about 4,000% increase, about 120% - about 3,000% increase, about 120% - about 2,000% increase, about 120% - about 1,000% increase, about 120% - about 800% increase, about 120% - about 600% increase, about 120% - about 500% increase, about 120% - about 400% increase, about 120% - about 300% increase, about 120% - about 200% increase, about 120% - about 180% increase, about 120% - about 160% increase, about 120% - about 140% increase, about 140% - about 5,000% increase, about 140% - about 4,000% increase, about 140% - about 3,000% increase, about 140% - about 2,000% increase, about 140% - about 1,000% increase, about 140% - about 800% increase, about 140% - about 600% increase, about 140% - about 500% increase, about 140% - about 400% increase, about 140% - about 300% increase, about 140% - about 200% increase, about 140% - about 180% increase, about 140% - about 160% increase, about 160% - about 5,000% increase, about 160% - about 4,000% increase, about 160% - about 3,000% increase, about 160% - about 2,000% increase, about 160% - about 1,000% increase, about 160% - about 800% increase, about 160% - about 600% increase, about 160% - about 500% increase, about 160% - about 400% increase, about 160% - about 300% increase, about 160% - about 200% increase, about 160% - about 180% increase, about 180% - about 5,000% increase, about 180% - about 4,000% increase, about 180% - about 3,000% increase, about 180% - about 2,000% increase, about 180% - about 1,000% increase, about 180% - about 800% increase, about 180% - about 600% increase, about 180% - about 500% increase, about 180% - about 400% increase, about 180% - about 300% increase, about 180% - about 200% increase, about 200% - about 5,000% increase, about 200% - about 4,000% increase, about 200% - about 3,000% increase, about 200% - about 2,000% increase, about 200% - about 1,An increase of 000%, an increase of about 200% to about 800%, an increase of about 200% to about 600%, an increase of about 200% to about 500%, an increase of about 200% to about 400%, an increase of about 200% to about 300%, an increase of about 300% to about 5,000%, an increase of about 300% to about 4,000%, an increase of about 300% to about 3,000%, an increase of about 300% to about 2,000%, an increase of about 300% to about 1,000%, an increase of about 300% to about 800%, an increase of about 300% to about 600%, an increase of about 300% to about 500%, an increase of about 300% to about 400%, an increase of about 400% to about 5,000%, an increase of about 400% to about 4,000%, an increase of about 400% to about 3,000%, an increase of about 400% to about 2,000%, an increase of about 400% to about 1,000%, an increase of about 400% to about 800%, an increase of about 400% to about 600%, an increase of about 400% to about 500%, an increase of about 500% to about 5,000%, an increase of about 500% to about 4,000%, an increase of about 500% to about 3,000%, an increase of about 500% to about 2,000%, an increase of about 500% to about 1,000%, an increase of about 500% to about 800%, an increase of about 500% to about 600%, an increase of about 600% to about 5,000%, an increase of about 600% to about 4,000%, an increase of about 600% to about 3,000%, an increase of about 600% to about 2,000%, an increase of about 600% to about 1,000%, an increase of about 600% to about 800%, an increase of about 800% to about 5,000%, an increase of about 800% to about 4,000%, an increase of about 800% to about 3,000%, an increase of about 800% to about 2,000%, an increase of about 800% to about 1,000%, an increase of about 1,000% to about 5,000%, an increase of about 1,000% to about 4,000%, an increase of about 1,000% to about 3,000%, an increase of about 1,000% to about 2,000%, an increase of about 2,000% to about 5,000%, an increase of about 2,000% to about 4,000%, an increase of about 2,000% to about 3,000%, an increase of about 3,000% to about 5,000%, an increase of about 3,000% to about 4,000%, or an increase of about 4,000% to about 5,000% is brought about.,

[0037] In some embodiments of any of the methods described herein, by this method, compared to the number of NK cells before the step of contacting the NK cells with any of the exemplary first multi-chain chimeric polypeptides described herein, for example, about 5-fold to about 5,000-fold, about 5-fold to about 4,500-fold, about 5-fold to about 4,000-fold, about 5-fold to about 3,500-fold, about 5-fold to about 3,000-fold, about 5-fold to about 2,500-fold, about 5-fold to about 2,000-fold, about 5-fold to about 1,500-fold, about 5-fold to about 1,000-fold, about 5-fold to about 800-fold, about 5-fold to about 600-fold, about 5-fold to about 500-fold, about 5-fold to about 450-fold, about 5-fold to about 400-fold, about 5-fold to about 350-fold, about 5-fold to about 300-fold, about 5-fold to about 250-fold, about 5-fold to about 200-fold, about 5-fold to about 150-fold, about 5-fold to about 100-fold, about 5-fold to about 80-fold, about 5-fold to about 60-fold, about 5-fold to about 40-fold, about 5-fold to about 20-fold, about 5-fold to about 10-fold, about 10-fold to about 5,000-fold, about 10-fold to about 4,500-fold, about 10-fold to about 4,000-fold, about 10-fold to about 3,500-fold, about 10-fold to about 3,000-fold, about 10-fold to about 2,500-fold, about 10-fold to about 2,000-fold, about 10-fold to about 1,500-fold, about 10-fold to about 1,000-fold, about 10-fold to about 800-fold, about 10-fold to about 600-fold, about 10-fold to about 500-fold, about 10-fold to about 450-fold, about 10-fold to about 400-fold, about 10-fold to about 350-fold, about 10-fold to about 300-fold, about 10-fold to about 250-fold, about 10-fold to about 200-fold, about 10-fold to about 150-fold, about 10-fold to about 100-fold, about 10-fold to about 80-fold, about 10-fold to about 60-fold, about 10-fold to about 40-fold, about 10-fold to about 20-fold, about 20-fold to about 5,000-fold, about 20-fold to about 4,500-fold, about 20-fold to about 4,000-fold, about 20-fold to about 3,500-fold, about 20-fold to about 3,000-fold, about 20-fold to about 2,500-fold, about 20-fold to about 2,000-fold, about 20-fold to about 1,500-fold, about 20-fold to about 1,000-fold, about 20-fold to about 800-fold, about 20-fold to about 600-fold, about 20-fold to about 500-fold, about 20-fold to about 450-fold, about 20-fold to about 400-fold, about 20-fold to about 350-fold, about 20-fold to about 300-fold, about 20-fold to about 250-fold, about 20-fold to about 200-fold, about 20-fold to about 150-fold, about 20-fold to about 100-fold, about 20-fold to about 80-fold, about 20-fold to about 60-fold, about 20-fold to about 40-fold, about 40-fold to about 5,000-fold, about 40-fold to about 4,500-fold, about 40-fold to about 4,000-fold, about 40-fold to about 3,500-fold, about 40-fold to about 3,000-fold, about 40-fold to about 2,500-fold, about 40-fold to about 2,000 times, about 40 times to about 1,500 times, about 40 times to about 1,000 times, about 40 times to about 800 times, about 40 times to about 600 times, about 40 times to about 500 times, about 40 times to about 450 times, about 40 times to about 400 times, about 40 times to about 350 times, about 40 times to about 300 times, about 40 times to about 250 times, about 40 times to about 200 times, about 40 times to about 150 times, about 40 times to about 100 times, about 40 times to about 80 times, about 40 times to about 60 times, about 60 times to about 5,000 times, about 60 times to about 4,500 times, about 60 times to about 4,000 times, about 60 times to about 3,500 times, about 60 times to about 3,000 times, about 60 times to about 2,500 times, about 60 times to about 2,000 times, about 60 times to about 1,500 times, about 60 times to about 1,000 times, about 60 times to about 800 times, about 60 times to about 600 times, about 60 times to about 500 times, about 60 times to about 450 times, about 60 times to about 400 times, about 60 times to about 350 times, about 60 times to about 300 times, about 60 times to about 250 times, about 60 times to about 200 times, about 60 times to about 150 times, about 60 times to about 100 times, about 60 times to about 80 times, about 80 times to about 5,000 times, about 80 times to about 4,500 times, about 80 times to about 4,000 times, about 80 times to about 3,500 times, about 80 times to about 3,000 times, about 80 times to about 2,500 times, about 80 times to about 2,000 times, about 80 times to about 1,500 times, about 80 times to about 1,000 times, about 80 times to about 800 times, about 80 times to about 600 times, about 80 times to about 500 times, about 80 times to about 450 times, about 80 times to about 400 times, about 80 times to about 350 times, about 80 times to about 300 times, about 80 times to about 250 times, about 80 times to about 200 times, about 80 times to about 150 times, about 80 times to about 100 times, about 100 times to about 5,000 times, about 100 times to about 4,500 times, about 100 times to about 4,000 times, about 100 times to about 3,500 times, about 100 times to about 3,000 times, about 100 times to about 2,500 times, about 100 times to about 2,000 times, about 100 times to about 1,500 times, about 100 times to about 1,000 times, about 100 times to about 800 times, about 100 times to about 600 times, about 100 times to about 500 times, about 100 times to about 450 times, about 100 times to about 400 times, about 100 times to about 350 times, about 100 times to about 300 times, about 100 times to about 250 times, about 100 times to about 200 times, about 100 times to about 150 times, about 150 times to about 5,000 times, about 150 times to about 4,500 times, about 150 times to about 4,000 times, about 150 times to about 3,500 times, about 150 times to about 3,000 times, about 150 times to about 2,500 times, about 150 times to about 2,000 times, about 150 times to about 1,500 times, about 150 times to about 1,000 times, about 150 times to about 800 times, about 150 times to about 600 times, about 150 times to about 500 times, about 150 times to about 450 times, about 150 times to about 400 times, about 150 times to about 350 times, about 150 times to about 300 times, about 150 times to about 250 times, about 150 times to about 200 times, about 200 times to about 5,000 times, about 200 times to about 4,500 times, about 200 times to about 4,000 times, about 200 times to about 3,500 times, about 200 times to about 3,000 times, about 200 times to about 2,500 times, about 200 times to about 2,000 times, about 200 times to about 1,500 times, about 200 times to about 1,000 times, about 200 times to about 800 times, about 200 times to about 600 times, about 200 times to about 500 times, about 200 times to about 450 times, about 200 times to about 400 times, about 200 times to about 350 times, about 200 times to about 300 times, about 200 times to about 250 times, about 250 times to about 5,000 times, about 250 times to about 4,500 times, about 250 times to about 4,000 times, about 250 times to about 3,500 times, about 250 times to about 3,000 times, about 250 times to about 2,500 times, about 250 times to about 2,000 times, about 250 times to about 1,500 times, about 250 times to about 1,000 times, about 250 times to about 800 times, about 250 times to about 600 times, about 250 times to about 500 times, about 250 times to about 450 times, about 250 times to about 400 times, about 250 times to about 350 times, about 250 times to about 300 times, about 300 times to about 5,000 times, about 300 times to about 4,500 times, about 300 times to about 4,000 times, about 300 times to about 3,500 times, about 300 times to about 3,000 times, about 300 times to about 2,500 times, about 300 times to about 2,000 times, about 300 times to about 1,500 times, about 300 times to about 1,000 times, about 300 times to about 800 times, about 300 times to about 600 times, about 300 times to about 500 times, about 300 times to about 450 times, about 300 times to about 400 times, about 300 times to about 350 times, about 350 times to about 5,000 times, about 350 times to about 4,500 times, about 350 times to about 4,000 times, about 350 times to about 3,500 times, about 350 times to about 3,000 times, about 350 times to about 2,500 times, about 350 times to about 2,000 times, about 350 times to about 1,500 times, about 350 times to about 1,000 times, about 350 times to about 800 times, about 350 times to about 600 times, about 350 times to about 500 times, about 350 times to about 450 times, about 350 times to about 400 times, about 400 times to about 5,000 times, about 400 times to about 4,500 times, about 400 times to about 4,000 times, about 400 times to about 3,500 times, about 400 times to about 3,000 times, about 400 times to about 2,500 times, about 400 times to about 2,000 times, about 400 times to about 1,500 times, about 400 times to about 1,000 times, about 400 times to about 800 times, about 400 times to about 600 times, about 400 times to about 500 times, about 400 times to about 450 times, about 450 times to about 5,000 times, about 450 times to about 4,500 times, about 450 times to about 4,000 times, about 450 times to about 3,500 times, about 450 times to about 3,000 times, about 450 times to about 2,500 times, about 450 times to about 2,000 times, about 450 times to about 1,500 times, about 450 times to about 1,000 times, about 450 times to about 800 times, about 450 times to about 600 times, about 450 times to about 500 times, about 500 times to about 5,000 times, about 500 times to about 4,500 times, about 500 times to about 4,000 times, about 500 times to about 3,500 times, about 500 times to about 3,000 times, about 500 times to about 2,500 times, about 500 times to about 2,000 times, about 500 times to about 1,500 times, about 500 times to about 1,000 times, about 500 times to about 800 times, about 500 times to about 600 times, about 600 times to about 5,000 times, about 600 times to about 4,500 times, about 600 times to about 4,000 times, about 600 times to about 3,500 times, about 600 times to about 3,000 times, about 600 times to about 2,500 times, about 600 times to about 2,000 times, about 600 times to about 1,500 times, about 600 times to about 1,000 times, about 600 times to about 800 times, about 800 times to about 5,000 times, about 800 times to about 4,500 times, about 800 times to about 4,000 times, about 800 times to about 3,500 times, about 800 times to about 3,000 times, about 800 times to about 2,500 times, about 800 times to about 2,000 times, about 800 times to about 1,500 times, about 800 times to about 1,000 times, about 1,000 times to about 5,000 times, about 1,000 times to about 4,500 times, about 1,000 times to about 4,000 times, about 1,000 times to about 3,500 times, about 1,000 times to about 3,000 times, about 1,000 times to about 2,500 times, about 1,000 times to about 2,000 times, about 1,000 times to about 1,500 times, about 1,500 times to about 5,000 times, about 1,500 times to about 4,500 times, about 1,500 times to about 4,000 times, about 1,500 times to about 3,500 times, about 1,500 times to about 3,000 times, about 1,500 times to about 2,500 times, about 1,500 times to about 2,000 times, about 2,000 times to about 5,000 times, about 2,000 times to about 4,500 times, about 2,000 times to about 4,Multiplication by 1000-fold, approximately 2000-fold to approximately 3500-fold, approximately 2000-fold to approximately 3000-fold, approximately 2000-fold to approximately 2500-fold, approximately 2500-fold to approximately 5000-fold, approximately 2500-fold to approximately 4500-fold, approximately 2500-fold to approximately 4000-fold, approximately 2500-fold to approximately 3500-fold, approximately 2500-fold to approximately 3000-fold, approximately 3000-fold to approximately 5000-fold, approximately 3000-fold to approximately 4500-fold, approximately 3000-fold to approximately 4000-fold, approximately 3000-fold to approximately 3500-fold, approximately 3500-fold to approximately 5000-fold, approximately 3500-fold to approximately 4500-fold, approximately 3500-fold to approximately 4000-fold, approximately 4000-fold to approximately 5000-fold, approximately 4000-fold to approximately 4500-fold, or approximately 4500-fold to approximately 5000-fold results in the proliferation of viable NK cells.,

[0038] In some embodiments, the method provides NK cells having improved persistence and cytotoxicity against diseased cells in a subject as compared to untreated control NK cells.,

[0039] Multichain chimeric polypeptide The methods described herein include the use of two different multichain chimeric polypeptides.,

[0040] The first multichain chimeric polypeptide provided herein includes (a) a first chimeric polypeptide comprising a first target binding domain, a soluble tissue factor domain, and a first domain of an affinity domain pair, and (b) a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair, and the first target binding domain and the second target binding domain each independently specifically bind to the IL-18 receptor or the IL-12 receptor.,

[0041] The second multichain chimeric polypeptide provided herein comprises: (a) a first chimeric polypeptide comprising a first target binding domain, a linker domain, and a first domain of an affinity domain pair; and (b) a second chimeric polypeptide comprising a second domain of the affinity domain pair and a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair, and the first target binding domain and the second target binding domain each independently specifically bind to the IL-7 receptor or the IL-21 receptor.

[0042] In some examples of either the first and second multichain chimeric polypeptides described herein, the full lengths of the first chimeric polypeptide and / or the second chimeric polypeptide are each independently from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, from about 50 amino acids to about 700 amino acids, from about 50 amino acids to about 650 amino acids, from about 50 amino acids to about 600 amino acids, from about 50 amino acids to about 550 amino acids, from about 50 amino acids to about 500 amino acids, from about 50 amino acids to about 480 amino acids, from about 50 amino acids to about 460 amino acids, from about 50 amino acids to about 440 amino acids, from about 50 amino acids to about 420 amino acids, from about 50 amino acids to about 400 amino acids, from about 50 amino acids to about 380 amino acids, from about 50 amino acids to about 360 amino acids, from about 50 amino acids to about 340 amino acids, from about 50 amino acids to about 320 amino acids, from about 50 amino acids to about 300 amino acids, from about 50 amino acids to about 280 amino acids, from about 50 amino acids to about 260 amino acids, from about 50 amino acids to about 240 amino acids, from about 50 amino acids to about 220 amino acids, from about 50 amino acids to about 200 amino acids, from about 50 amino acids to about 150 amino acids, from about 50 amino acids to about 100 amino acids, from about 100 amino acids to about 3000 amino acids, from about 100 amino acids to about 2500 amino acids, from about 100 amino acids to about 2000 amino acids, from about 100 amino acids to about 1500 amino acids, from about 100 amino acids to about 1000 amino acids, from about 100 amino acids to about 950 amino acids, from about 100 amino acids to about 900 amino acids, from about 100 amino acids to about 850 amino acids, from about 100 amino acids to about 800 amino acids, from about 100 amino acids to about 750 amino acids, from about 100 amino acids to about 700 amino acids, from about 100 amino acids to about 650 amino acids, from about 100 amino acids to about 600 amino acids, from about 100 amino acids to about 550 amino acids, from about 100 amino acids to about 500 amino acids, from about 100 amino acids to about 480 amino acids, from about 100 amino acids to about 460 amino acids, from about 100 amino acids to about 440 amino acids, from about 100 amino acids to about 420 amino acids, from about 100 amino acids to about 400 amino acids,From about 100 amino acids to about 380 amino acids, from about 100 amino acids to about 360 amino acids, from about 100 amino acids to about 340 amino acids, from about 100 amino acids to about 320 amino acids, from about 100 amino acids to about 300 amino acids, from about 100 amino acids to about 280 amino acids, from about 100 amino acids to about 260 amino acids, from about 100 amino acids to about 240 amino acids, from about 100 amino acids to about 220 amino acids, from about 100 amino acids to about 200 amino acids, from about 100 amino acids to about 150 amino acids, from about 150 amino acids to about 3000 amino acids, from about 150 amino acids to about 2500 amino acids, from about 150 amino acids to about 2000 amino acids, from about 150 amino acids to about 1500 amino acids, from about 150 amino acids to about 1000 amino acids, from about 150 amino acids to about 950 amino acids, from about 150 amino acids to about 900 amino acids, from about 150 amino acids to about 850 amino acids, from about 150 amino acids to about 800 amino acids, from about 150 amino acids to about 750 amino acids, from about 150 amino acids to about 700 amino acids, from about 150 amino acids to about 650 amino acids, from about 150 amino acids to about 600 amino acids, from about 150 amino acids to about 550 amino acids, from about 150 amino acids to about 500 amino acids, from about 150 amino acids to about 480 amino acids, from about 150 amino acids to about 460 amino acids, from about 150 amino acids to about 440 amino acids, from about 150 amino acids to about 420 amino acids, from about 150 amino acids to about 400 amino acids, from about 150 amino acids to about 380 amino acids, from about 150 amino acids to about 360 amino acids, from about 150 amino acids to about 340 amino acids, from about 150 amino acids to about 320 amino acids, from about 150 amino acids to about 300 amino acids, from about 150 amino acids to about 280 amino acids, from about 150 amino acids to about 260 amino acids, from about 150 amino acids to about 240 amino acids, from about 150 amino acids to about 220 amino acids, from about 150 amino acids to about 200 amino acids, from about 200 amino acids to about 3000 amino acids, from about 200 amino acids to about 2500 amino acids, from about 200 amino acids to about 2000 amino acids, from about 200 amino acids to about 1500 amino acids, from about 200 amino acids to about 1000 amino acids, from about 200 amino acids to about 950 amino acids, from about 200 amino acids to about 900 amino acids, from about 200 amino acids to about 850 amino acids, from about 200 amino acids to about 800 amino acids, from about 200 amino acids to about 750 amino acids, from about 200 amino acids to about 700 amino acids, from about 200 amino acids to about 650 amino acids, from about 200 amino acids to about 600 amino acids, from about 200 amino acids to about 550 amino acids,About 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids, about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids,From about 240 to about 600 amino acids, from about 240 to about 550 amino acids, from about 240 to about 500 amino acids, from about 240 to about 480 amino acids, from about 240 to about 460 amino acids, from about 240 to about 440 amino acids, from about 240 to about 420 amino acids, from about 240 to about 400 amino acids, from about 240 to about 380 amino acids, from about 240 to about 360 amino acids, from about 240 to about 340 amino acids, from about 240 to about 320 amino acids, from about 240 to about 300 amino acids, from about 240 to about 280 amino acids, from about 260 to about 3000 amino acids, from about 260 to about 2500 amino acids, from about 260 to about 2000 amino acids, from about 260 to about 1500 amino acids, from about 260 to about 1000 amino acids, from about 260 to about 950 amino acids, from about 260 to about 900 amino acids, from about 260 to about 850 amino acids, from about 260 to about 800 amino acids, from about 260 to about 750 amino acids, from about 260 to about 700 amino acids, from about 260 to about 650 amino acids, from about 260 to about 600 amino acids, from about 260 to about 550 amino acids, from about 260 to about 500 amino acids, from about 260 to about 480 amino acids, from about 260 to about 460 amino acids, from about 260 to about 440 amino acids, from about 260 to about 420 amino acids, from about 260 to about 400 amino acids, from about 260 to about 380 amino acids, from about 260 to about 360 amino acids, from about 260 to about 340 amino acids, from about 260 to about 320 amino acids, from about 260 to about 300 amino acids, from about 260 to about 280 amino acids, from about 280 to about 3000 amino acids, from about 280 to about 2500 amino acids, from about 280 to about 2000 amino acids, from about 280 to about 1500 amino acids, from about 280 to about 1000 amino acids, from about 280 to about 950 amino acids, from about 280 to about 900 amino acids, from about 280 to about 850 amino acids, from about 280 to about 800 amino acids, from about 280 to about 750 amino acids, from about 280 to about 700 amino acids, from about 280 to about 650 amino acids, from about 280 to about 600 amino acids, from about 280 to about 550 amino acidsAbout 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids, about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acidsFrom about 320 amino acids to about 380 amino acids, from about 320 amino acids to about 360 amino acids, from about 320 amino acids to about 340 amino acids, from about 340 amino acids to about 3000 amino acids, from about 340 amino acids to about 2500 amino acids, from about 340 amino acids to about 2000 amino acids, about 340 amino acids ~about 1500 amino acids, about 340 amino acids~about 1000 amino acids, about 340 amino acids~about 950 amino acids, about 340 amino acids~about 900 amino acids, about 340 amino acids~about 850 amino acids, about 340 amino acids~about 800 amino acids, about 340 amino acids~about 750 amino acids, about 340 amino acids~about 700 amino acids, about 340 amino acids~about 650 amino acids, about 340 amino acids~about 600 amino acids, about 340 amino acids~about 550 amino acids, about 340 amino acids~about 500 amino acids, about 340 amino acids~about 480 amino acids, about 340 amino acids~about 460 amino acids, about 340 amino acids~about 440 amino acids, about 340 amino acids~about 420 amino acids, about 340 amino acids~about 400 amino acids, about 340 amino acids~about 380 amino acids, about 340 amino acids~about 360 amino acids, about 360 amino acids~about 3000 amino acids, about 360 amino acids~about 2500 amino acids, about 360 amino acids~about 2000 amino acids, about 360 amino acids~about 1500 amino acids, about 360 amino acids~about 1000 amino acids, about 360 amino acids~about 950 amino acids, about 360 amino acids~about 900 amino acids, about 360 amino acids~about 850 amino acids, about 360 amino acids~about 800 amino acids, about 360 amino acids~about 750 amino acids, about 360 amino acids~about 700 amino acids, about 360 amino acids~about 650 amino acids, about 360 amino acids~about 600 amino acids, about 360 amino acids~about 550 amino acids, about 360 amino acids~about 500 amino acids, about 360 amino acids~about 480 amino acids, about 360 amino acids~about 460 amino acids, about 360 amino acids~about 440 amino acids, about 360 amino acids~about 420 amino acids, about 360 amino acids~about 400 amino acids, about 360 amino acids~about 380 amino acids, about 380 amino acids~about 3000 amino acids, about 380 amino acids~about 2500 amino acids, about 380 amino acids~about 2000 amino acids, about 380 amino acids~about 1500 amino acids, about 380 amino acids~about 1000 amino acids, about 380 amino acids~about 950 amino acids, about 380 amino acids~about 900 amino acids, about 380 amino acids~about 850 amino acids, about 380 amino acids~about 800 amino acids, about 380 amino acids~about 750 amino acids, about 380 amino acids~about 700 amino acids, about 380 amino acids~about 650 amino acids, about 380 amino acids~about 600 amino acids, about 380 amino acids~about 550 amino acids, about 380 amino acids~about 500 amino acids,From about 380 amino acids to about 480 amino acids, from about 380 amino acids to about 460 amino acids, from about 380 amino acids to about 440 amino acids, from about 380 amino acids to about 420 amino acids, from about 380 amino acids to about 400 amino acids, from about 400 amino acids to about 3000 amino acids, from about 400 amino acids to about 2500 amino acids, from about 400 amino acids to about 2000 amino acids, from about 400 amino acids to about 1500 amino acids, from about 400 amino acids to about 1000 amino acids, from about 400 amino acids to about 950 amino acids, from about 400 amino acids to about 900 amino acids, from about 400 amino acids to about 850 amino acids, from about 400 amino acids to about 800 amino acids, from about 400 amino acids to about 750 amino acids, from about 400 amino acids to about 700 amino acids, from about 400 amino acids to about 650 amino acids, from about 400 amino acids to about 600 amino acids, from about 400 amino acids to about 550 amino acids, from about 400 amino acids to about 500 amino acids, from about 400 amino acids to about 480 amino acids, from about 400 amino acids to about 460 amino acids, from about 400 amino acids to about 440 amino acids, from about 400 amino acids to about 420 amino acids, from about 420 amino acids to about 3000 amino acids, from about 420 amino acids to about 2500 amino acids, from about 420 amino acids to about 2000 amino acids, from about 420 amino acids to about 1500 amino acids, from about 420 amino acids to about 1000 amino acids, from about 420 amino acids to about 950 amino acids, from about 420 amino acids to about 900 amino acids, from about 420 amino acids to about 850 amino acids, from about 420 amino acids to about 800 amino acids, from about 420 amino acids to about 750 amino acids, from about 420 amino acids to about 700 amino acids, from about 420 amino acids to about 650 amino acids, from about 420 amino acids to about 600 amino acids, from about 420 amino acids to about 550 amino acids, from about 420 amino acids to about 500 amino acids, from about 420 amino acids to about 480 amino acids, from about 420 amino acids to about 460 amino acids, from about 420 amino acids to about 440 amino acids, from about 440 amino acids to about 3000 amino acids, from about 440 amino acids to about 2500 amino acids, from about 440 amino acids to about 2000 amino acids, from about 440 amino acids to about 1500 amino acids, from about 440 amino acids to about 1000 amino acids, from about 440 amino acids to about 950 amino acids, from about 440 amino acids to about 900 amino acids, from about 440 amino acids to about 850 amino acids, from about 440 amino acids to about 800 amino acids, from about 440 amino acids to about 750 amino acids, from about 440 amino acids to about 700 amino acids, from about 440 amino acids to about 650 amino acids,From about 440 amino acids to about 600 amino acids, from about 440 amino acids to about 550 amino acids, from about 440 amino acids to about 500 amino acids, from about 440 amino acids to about 480 amino acids, from about 440 amino acids to about 460 amino acids, from about 460 amino acids to about 3000 amino acids, from about 460 amino acids to about 2500 amino acids, from about 460 amino acids to about 2000 amino acids, from about 460 amino acids to about 1500 amino acids, from about 460 amino acids to about 1000 amino acids, from about 460 amino acids to about 950 amino acids, from about 460 amino acids to about 900 amino acids, from about 460 amino acids to about 850 amino acids, from about 460 amino acids to about 800 amino acids, from about 460 amino acids to about 750 amino acids, from about 460 amino acids to about 700 amino acids, from about 460 amino acids to about 650 amino acids, from about 460 amino acids to about 600 amino acids, from about 460 amino acids to about 550 amino acids, from about 460 amino acids to about 500 amino acids, from about 460 amino acids to about 480 amino acids, from about 480 amino acids to about 3000 amino acids, from about 480 amino acids to about 2500 amino acids, from about 480 amino acids to about 2000 amino acids, from about 480 amino acids to about 1500 amino acids, from about 480 amino acids to about 1000 amino acids, from about 480 amino acids to about 950 amino acids, from about 480 amino acids to about 900 amino acids, from about 480 amino acids to about 850 amino acids, from about 480 amino acids to about 800 amino acids, from about 480 amino acids to about 750 amino acids, from about 480 amino acids to about 700 amino acids, from about 480 amino acids to about 650 amino acids, from about 480 amino acids to about 600 amino acids, from about 480 amino acids to about 550 amino acids, from about 480 amino acids to about 500 amino acids, from about 500 amino acids to about 3000 amino acids, from about 500 amino acids to about 2500 amino acids, from about 500 amino acids to about 2000 amino acids, from about 500 amino acids to about 1500 amino acids, from about 500 amino acids to about 1000 amino acids, from about 500 amino acids to about 950 amino acids, from about 500 amino acids to about 900 amino acids, from about 500 amino acids to about 850 amino acids, from about 500 amino acids to about 800 amino acids, from about 500 amino acids to about 750 amino acids, from about 500 amino acids to about 700 amino acids, from about 500 amino acids to about 650 amino acids, from about 500 amino acids to about 600 amino acids, from about 500 amino acids to about 550 amino acids, from about 550 amino acids to about 3000 amino acids, from about 550 amino acids to about 2500 amino acids, from about 550 amino acids to about 2000 amino acids, from about 550 amino acids to about 1500 amino acids,About 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids,from about 800 amino acids to about 1500 amino acids, from about 800 amino acids to about 1000 amino acids, from about 800 amino acids to about 950 amino acids, from about 800 amino acids to about 900 amino acids, from about 800 amino acids to about 850 amino acids, from about 850 amino acids to about 3000 amino acids, from about 850 amino acids to about 2500 amino acids, from about 850 amino acids to about 2000 amino acids, from about 850 amino acids to about 1500 amino acids, from about 850 amino acids to about 1000 amino acids, from about 850 amino acids to about 950 amino acids, from about 850 amino acids to about 900 amino acids, from about 900 amino acids to about 3000 amino acids, from about 900 amino acids to about 2500 amino acids, from about 900 amino acids to about 2000 amino acids, from about 900 amino acids to about 1500 amino acids, from about 900 amino acids to about 1000 amino acids, from about 900 amino acids to about 950 amino acids, from about 950 amino acids to about 3000 amino acids, from about 950 amino acids to about 2500 amino acids, from about 950 amino acids to about 2000 amino acids, from about 950 amino acids to about 1500 amino acids, from about 950 amino acids to about 1000 amino acids, from about 1000 amino acids to about 3000 amino acids, from about 1000 amino acids to about 2500 amino acids, from about 1000 amino acids to about 2000 amino acids, from about 1000 amino acids to about 1500 amino acids, from about 1500 amino acids to about 3000 amino acids, from about 1500 amino acids to about 2500 amino acids, from about 1500 amino acids to about 2000 amino acids, from about 2000 amino acids to about 3000 amino acids, from about 2000 amino acids to about 2500 amino acids, or from about 2500 amino acids to about 3000 amino acids. Schematic diagrams of exemplary first multi-chain chimeric polypeptides provided herein are shown in FIGS. 1-4. Schematic diagrams of exemplary second multi-chain chimeric polypeptides are shown in FIGS. 5-12.,

[0043] In some embodiments of either the first and second multichain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the linker domain (e.g., any of the exemplary linker domains described herein) are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of either the first and second multichain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the linker domain (e.g., any of the exemplary linker domains described herein) within the first chimeric polypeptide.

[0044] In some embodiments of either the first and second multichain chimeric polypeptides described herein, the linker domain (e.g., any of the exemplary linker domains described herein) and the first domain of the affinity domain pair (e.g., any of the exemplary first domains of any of the exemplary affinity domain pairs described herein) are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of either the first and second multichain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the linker domain (e.g., any of the exemplary linker domains described herein) and the first domain of the affinity domain pair (e.g., any of the exemplary first domains of any of the exemplary affinity domain pairs described herein) within the first chimeric polypeptide.

[0045] In some embodiments of either the first and second multichain chimeric polypeptides described herein, the second domain of the affinity domain pair (e.g., any of the exemplary second domains of any of the exemplary affinity domain pairs described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of either the first and second multichain chimeric polypeptides described herein, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of the affinity domain pair (e.g., any of the exemplary second domains of any of the exemplary affinity domain pairs described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) within the second chimeric polypeptide.

[0046] Tissue factor Human tissue factor is a 263 amino acid transmembrane protein ((UniProtKB identifier number: P13726) that includes the following 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). The cytoplasmic tail contains two phosphorylation sites at Ser 253 and Ser 258 and one S-palmitoylation site at Cys 245 No deletion or mutation of the cytoplasmic domain has been found to affect the coagulation activity of tissue factor. Tissue factor has one S-palmitoylation site at Cys 245 within the intracellular domain of the protein. Cys 245 is located at the amino terminus of the intracellular domain and is near the membrane surface. The tissue factor transmembrane domain is composed of a single-spanning α-helix.

[0047] The extracellular domain of tissue factor, which consists of two fibronectin type III domains, is linked to the transmembrane domain via a six-amino acid linker. This linker provides conformational mobility to separate the extracellular domain of tissue from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping β-sheets, with the upper sheet domain containing three antiparallel β-strands and the lower sheet containing four β-strands. The β-strands are linked by β-loops between strands βA and βB, βC and βD, and βE and βF, and all of these conformations are conserved within the two modules. There are three short α-helix segments that link the β-strands. A tissue-specific feature is a 17-amino acid β-hairpin between strand β10 and strand β11, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is absent from the C-terminal domain and is specific to tissue factor. Such a fibronectin type III domain structure is characteristic of the immunoglobulin-like protein family fold and is conserved among a wide variety of extracellular proteins.

[0048] Thrombinogen FVII is rapidly converted to FVIIa by proteolytic cleavage when it binds to tissue to form the active tissue factor-FVIIa complex. FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. The allosteric interaction between tissue and FVIIa on the tissue factor-FVIIa complex greatly increases the enzymatic activity of FVIIa, increasing the hydrolysis rate of a small chromogenic peptidyl substrate by approximately 20 - 100-fold and increasing the activation rate of the natural macromolecular substrates FIX and FX by nearly one million-fold. In concert with the allosteric activation of the active site of FVIIa upon binding to tissue factor, the formation of the tissue-FVIIa complex on the phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface) is Ca 2+Increase the activation rate of FIX or FX by a further 1,000-fold in a dependent manner. The overall increase of approximately 1 million-fold in FX activation by tissue-FVIIa-lipid complex compared to free FVIIa is a critical regulatory point of the coagulation cascade.

[0049] FVII is a single-chain polypeptide of approximately 50 kDa, consisting of 406 amino acid residues, and has an N-terminal γ-carboxyglutamic acid-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII is activated to FVIIa by specific proteolytic cleavage of the bond at Ile- 154 -Arg 152 This cleavage causes the light and heavy chains to be held together by a single disulfide bond between Cys 135 and Cys 262 FVIIa binds to the phospholipid membrane in a Ca 2+ -dependent manner via its N-terminal GLA domain. Immediately C-terminal to the GLA domain, there are an aromatic stack and two EGF domains. The aromatic stack links the GLA domain to the EGF1 domain that binds to a single Ca 2+ ion. Occupation of this Ca 2+ -binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consists of His 193 , Asp 242 , and Ser 344 and the binding of a single Ca 2+ ion within the FVIIa protease domain is essential for its catalytic activity. The proteolytic activity of FVII to FVIIa liberates the newly formed amino terminus at Ile 153 which folds back and inserts into the activation pocket, Asp 343Forms a salt bridge with the carboxylate salt to generate an oxoanion hole. The formation of this salt bridge is essential for FVIIa activity. However, the formation of the oxoanion hole does not occur in free FVIIa during proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is hardly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

[0050] The tissue factor-mediated arrangement of the FVIIa active site on the membrane surface is important for FVIIa towards homologous substrates. Free FVIIa adopts a stable extended structure when its active site binds to a membrane with the site located approximately 80 Å above the membrane surface. When FVIIa binds to tissue factor, the FVa active site is repositioned approximately 6 Å closer to the membrane. This regulation can help in the proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLA domainless FVIIa, it has been shown that the active site is still positioned at a similar distance from the membrane, indicating that tissue factor can fully support the FVIIa active site arrangement even in the absence of FVIIa-membrane interaction. Additional data showed that tissue factor supports the full proteolytic activity of FVIIa as long as the tissue factor extracellular domain is tethered to the membrane surface in some way. However, by raising the active site of FVIIa more than 80 Å above the membrane surface, the ability of the tissue factor-FVIIa complex to activate FX was significantly reduced, while the tissue factor-FVIIa amidolytic activity was not reduced.

[0051] Alanine scanning mutagenesis has been used to evaluate the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitutions identified a limited number of residue positions where the alanine substitution caused a 5- to 10-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be sufficiently exposed to solvent in the crystal structure to be compatible with macromolecular ligand interactions. The FVIIa ligand binding site is located over a broad region at the boundary between two modules. In the C-module, the residues Arg 135 and Phe 140 provide independent contacts with FVIIa. Leu 133 is located at the base of the finger-like structure and is packed into the gap between the two modules. This provides continuity to a major cluster of important binding residues consisting of Lys 20 , Thr 60 , Asp 58 , and Ile 22 . Thr 60 is only partially exposed to solvent and does not make significant contact with the ligand, but may play a local structural role. The binding site extends onto the concave side of the intermodule angle that includes Glu 24 and Gln 110 , as well as the potentially more distal residue Val 207 . The binding region extends into the convex region formed by Asp58, Lys 48 , Lys 46 , Gln 37 , Asp 44 , and Trp 45 . Trp 45 and Asp 44 do not interact independently with FVIIa, and the mutational effect at the Trp 45 position is on the adjacent Asp 44 and Gln 37This shows that it can reflect the structural importance of this side chain for local packing of the side chain. The interaction region contains two surface-exposed aromatic residues, Phe, which form part of a hydrophobic cluster within the N-module. 76 and Tyr 78 and further includes.

[0052] Known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as certain protease-activated receptors. By mutation analysis, several residues have been identified that, when mutated, support complete FVIIa amidolytic activity towards small peptidyl substrates but lack the ability to support the activation of macromolecular substrates (i.e., FVII, FIX, and FX) (Ruf et al., J Biol Chem 267(31):22206-22210, 1992, Ruf et al., J Biol Chem 267(9):6375-6381, 1992, Huang et al., J Biol Chem 271(36):21752-21757, 1996, Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region of residues 159-165, and residues within or adjacent to this mobile loop, have been shown to be essential for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate-binding exosite region of tissue factor that is quite distant from the FVIIa active site. Substituting glycine residues with slightly bulkier alanine residues significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the mobility provided by glycine is essential for the loop of residues 159-165 for tissue factor macromolecular substrate recognition.

[0053] Residues Lys 165 and Lys 166 have also been demonstrated to be important for substrate recognition and binding. Mutating either of these residues to alanine significantly reduces the tissue factor cofactor function. Lys 165 and Lys 166 are oriented outwards from each other, Lys165 refers to FVIIa within most of the tissue factor-FVIIa structure, Lys 166 refers to the substrate-binding exosite region within the crystal structure. The putative salt bridge formation between Lys of FVIIa 165 and Gla of FVIIa 35 would support the concept that the interaction of the GLA domain of FVIIa with tissue factor modulates substrate recognition. These results suggest that the C-terminal portion of the tissue factor extracellular domain interacts directly with the GLA domains of FIX and FX, and possibly the adjacent EGF1 domain, and that the presence of the FVIIa GLA domain may modulate these interactions either directly or indirectly.

[0054] Exemplary linker domains and IgG1 antibody constructs In some examples of any of the methods, compositions, or kits described herein, the linker domain within the second multichain chimeric polypeptide can be, or can include, any antigen recognized by a cognate IgG1 antibody (e.g., a monoclonal antibody). IgG1 antibodies are distinguished from antibodies of other IgG classes by their constant regions, particularly within the hinge region and upper CH2 domain (see, e.g., Vadarsson et al., IgG Subclasses and Allotypes: From Structure to Effector Functions, Front Immunol., 5, Article 520, 2014). Human IgG1 is the only known IgG subclass that binds to human CD16a and activates signaling of human CD16a.

[0055] Any of a variety of linker domains can be used within the second multichain chimeric polypeptide provided herein. In certain embodiments, the linker domain is recognized by an antibody (e.g., an IgG1 antibody) or an antibody fragment. In some embodiments, the linker domain is the kappa light chain of an antibody recognized by a cognate anti-kappa light chain IgG1 antibody. In some embodiments, the linker domain is the lambda light chain of an antibody recognized by a cognate anti-lambda light chain human IgG1 antibody. A variety of kappa and lambda light chains are known in the art (see, e.g., Smith et al., Antigen Nature and Complexity Influence Human Antibody Light Chain Usage and Specificity, Vaccine, 34(25):2813-2820, 2016). In some embodiments, the linker domain is or comprises a human polypeptide (e.g., the human kappa light chain of an antibody or the human lambda light chain of an antibody) recognized by a cognate human IgG1 antibody (e.g., a monoclonal antibody). In some embodiments, the cognate human IgG1 antibody comprises at least two antigen-binding domains, each antigen-binding domain specifically binding to the linker domain. In some embodiments, the cognate human IgG1 antibody comprises at least two antigen-binding domains, and only a subset (e.g., one) of those antigen-binding domains specifically binds to the linker domain within the second multichain chimeric polypeptide. In some embodiments, the linker domain can be any of the soluble tissue factor domains described herein.

[0056] In some embodiments of any of the methods, compositions, and kits described herein, the IgG1 antibody construct can be an IgG1 antibody (e.g., a monoclonal or polyclonal IgG1 antibody that specifically binds to a linker domain). In some embodiments of any of the methods, compositions, and kits described herein, the IgG1 antibody construct can be an antibody or antibody fragment that includes an IgG1 Fc region (e.g., a human IgG1 Fc region) and specifically binds to a linker domain within a second multichain chimeric polypeptide. As is known in the art, the IgG1 Fc region binds to CD16a (FcR gamma III) (e.g., human CD16a) and induces its intracellular signaling. In some embodiments, the IgG1 antibody construct can be a single-chain or multichain polypeptide that includes an Fc region and specifically binds to a linker domain, and this Fc region can specifically bind to CD16a (FcR gamma III) (e.g., human CD16a) and can induce its intracellular signaling in natural killer cells (e.g., human natural killer cells). In some embodiments, the IgG1 antibody construct can be a single-chain or multichain polypeptide that includes an Fc region and specifically binds to a linker domain, and this Fc region includes a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, or at least 99% identical) to a wild-type IgG1 Fc domain (e.g., a wild-type human IgG1 Fc domain, e.g., SEQ ID NO: 1), can specifically bind to CD16a (FcR gamma III) (e.g., human CD16a), and can induce its intracellular signaling in natural killer cells (e.g., human natural killer cells).In some embodiments of any of the methods, compositions, or kits described herein, the IgG1 antibody construct can be an antibody or antibody fragment that specifically binds to a linker domain and comprises a non-IgG1 Fc region (e.g., an IgG2, IgG3, or IgG4 Fc region), wherein the non-IgG1 Fc region is modified (e.g., by substituting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the wild-type non-IgG1 Fc region) such that the non-IgG1 Fc region can bind to CD16a (FcR gamma III) (e.g., human CD16a) and induce its intracellular signaling in natural killer cells (e.g., human natural killer cells). In some embodiments of any of the methods, compositions, or kits described herein, the IgG1 antibody construct specifically binds to a linker domain within a second multichain chimeric polypeptide and comprises a non-human Fc region (e.g., an Fc region derived from a non-human antibody), wherein the non-human Fc region is modified (e.g., by substituting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in the wild-type non-human Fc region) such that the non-human Fc region can bind to human CD16a (human FcR gamma III) and induce its intracellular signaling in natural killer cells (e.g., human natural killer cells). TIFF0007713393000025.tif139137

[0057] In some embodiments, the linker domain is from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 160 amino acids, from about 20 amino acids to about 155 amino acids, from about 20 amino acids to about 150 amino acids, from about 20 amino acids to about 145 amino acids, from about 20 amino acids to about 140 amino acids, from about 20 amino acids to about 135 amino acids, from about 20 amino acids to about 130 amino acids, from about 20 amino acids to about 125 amino acids, from about 20 amino acids to about 120 amino acids, from about 20 amino acids to about 115 amino acids, from about 20 amino acids to about 110 amino acids, from about 20 amino acids to about 105 amino acids, from about 20 amino acids to about 100 amino acids, from about 20 amino acids to about 95 amino acids, from about 20 amino acids to about 90 amino acids, from about 20 amino acids to about 85 amino acids, from about 20 amino acids to about 80 amino acids, from about 20 amino acids to about 75 amino acids, from about 20 amino acids to about 70 amino acids, from about 20 amino acids to about 60 amino acids, from about 20 amino acids to about 50 amino acids, from about 20 amino acids to about 40 amino acids, from about 20 amino acids to about 30 amino acids, from about 30 amino acids to about 220 amino acids, from about 30 amino acids to about 215 amino acids, from about 30 amino acids to about 210 amino acids, from about 30 amino acids to about 205 amino acids, from about 30 amino acids to about 200 amino acids, from about 30 amino acids to about 195 amino acids, from about 30 amino acids to about 190 amino acids, from about 30 amino acids to about 185 amino acids, from about 30 amino acids to about 180 amino acids, from about 30 amino acids to about 175 amino acids, from about 30 amino acids to about 170 amino acids, from about 30 amino acids to about 165 amino acids, from about 30 amino acids to about 160 amino acids, from about 30 amino acids to about 155 amino acids, from about 30 amino acids to about 150 amino acids, from about 30 amino acids to about 145 amino acids, from about 30 amino acids to about 140 amino acids, from about 30 amino acids to about 135 amino acids, from about 30 amino acids to about 130 amino acids, from about 30 amino acids to about 125 amino acids, from about 30 amino acids to about 120 amino acids, from about 30 amino acids to about 115 amino acids, from about 30 amino acids to about 110 amino acids,From about 30 amino acids to about 105 amino acids, from about 30 amino acids to about 100 amino acids, from about 30 amino acids to about 95 amino acids, from about 30 amino acids to about 90 amino acids, from about 30 amino acids to about 85 amino acids, from about 30 amino acids to about 80 amino acids, from about 30 amino acids to about 75 amino acids, from about 30 amino acids to about 70 amino acids, from about 30 amino acids to about 60 amino acids, from about 30 amino acids to about 50 amino acids, from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 220 amino acids, from about 40 amino acids to about 215 amino acids, from about 40 amino acids to about 210 amino acids, from about 40 amino acids to about 205 amino acids, from about 40 amino acids to about 200 amino acids, from about 40 amino acids to about 195 amino acids, from about 40 amino acids to about 190 amino acids, from about 40 amino acids to about 185 amino acids, from about 40 amino acids to about 180 amino acids, from about 40 amino acids to about 175 amino acids, from about 40 amino acids to about 170 amino acids, from about 40 amino acids to about 165 amino acids, from about 40 amino acids to about 160 amino acids, from about 40 amino acids to about 155 amino acids, from about 40 amino acids to about 150 amino acids, from about 40 amino acids to about 145 amino acids, from about 40 amino acids to about 140 amino acids, from about 40 amino acids to about 135 amino acids, from about 40 amino acids to about 130 amino acids, from about 40 amino acids to about 125 amino acids, from about 40 amino acids to about 120 amino acids, from about 40 amino acids to about 115 amino acids, from about 40 amino acids to about 110 amino acids, from about 40 amino acids to about 105 amino acids, from about 40 amino acids to about 100 amino acids, from about 40 amino acids to about 95 amino acids, from about 40 amino acids to about 90 amino acids, from about 40 amino acids to about 85 amino acids, from about 40 amino acids to about 80 amino acids, from about 40 amino acids to about 75 amino acids, from about 40 amino acids to about 70 amino acids, from about 40 amino acids to about 60 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 220 amino acids, from about 50 amino acids to about 215 amino acids, from about 50 amino acids to about 210 amino acids, from about 50 amino acids to about 205 amino acids, from about 50 amino acids to about 200 amino acids, from about 50 amino acids to about 195 amino acids, from about 50 amino acids to about 190 amino acids, from about 50 amino acids to about 185 amino acids, from about 50 amino acids to about 180 amino acids, from about 50 amino acids to about 175 amino acids, from about 50 amino acids to about 170 amino acids, from about 50 amino acids to about 165 amino acids, from about 50 amino acids to about 160 amino acids, from about 50 amino acids to about 155 amino acids, from about 50 amino acids to about 150 amino acidsfrom about 50 amino acids to about 145 amino acids, from about 50 amino acids to about 140 amino acids, from about 50 amino acids to about 135 amino acids, from about 50 amino acids to about 130 amino acids, from about 50 amino acids to about 125 amino acids, from about 50 amino acids to about 120 amino acids, from about 50 amino acids to about 115 amino acids, from about 50 amino acids to about 110 amino acids, from about 50 amino acids to about 105 amino acids, from about 50 amino acids to about 100 amino acids, from about 50 amino acids to about 95 amino acids, from about 50 amino acids to about 90 amino acids, from about 50 amino acids to about 85 amino acids, from about 50 amino acids to about 80 amino acids, from about 50 amino acids to about 75 amino acids, from about 50 amino acids to about 70 amino acids, from about 50 amino acids to about 60 amino acids, from about 60 amino acids to about 220 amino acids, from about 60 amino acids to about 215 amino acids, from about 60 amino acids to about 210 amino acids, from about 60 amino acids to about 205 amino acids, from about 60 amino acids to about 200 amino acids, from about 60 amino acids to about 195 amino acids, from about 60 amino acids to about 190 amino acids, from about 60 amino acids to about 185 amino acids, from about 60 amino acids to about 180 amino acids, from about 60 amino acids to about 175 amino acids, from about 60 amino acids to about 170 amino acids, from about 60 amino acids to about 165 amino acids, from about 60 amino acids to about 160 amino acids, from about 60 amino acids to about 155 amino acids, from about 60 amino acids to about 150 amino acids, from about 60 amino acids to about 145 amino acids, from about 60 amino acids to about 140 amino acids, from about 60 amino acids to about 135 amino acids, from about 60 amino acids to about 130 amino acids, from about 60 amino acids to about 125 amino acids, from about 60 amino acids to about 120 amino acids, from about 60 amino acids to about 115 amino acids, from about 60 amino acids to about 110 amino acids, from about 60 amino acids to about 105 amino acids, from about 60 amino acids to about 100 amino acids, from about 60 amino acids to about 95 amino acids, from about 60 amino acids to about 90 amino acids, from about 60 amino acids to about 85 amino acids, from about 60 amino acids to about 80 amino acids, from about 60 amino acids to about 75 amino acids, from about 60 amino acids to about 70 amino acids, from about 70 amino acids to about 220 amino acids, from about 70 amino acids to about 215 amino acids, from about 70 amino acids to about 210 amino acids, from about 70 amino acids to about 205 amino acids, from about 70 amino acids to about 200 amino acids, from about 70 amino acids to about 195 amino acids, from about 70 amino acids to about 190 amino acids, from about 70 amino acids to about 185 amino acids, from about 70 amino acids to about 180 amino acids, from about 70 amino acids to about 175 amino acids, from about 70 amino acids to about 170 amino acidsfrom about 70 amino acids to about 165 amino acids, from about 70 amino acids to about 160 amino acids, from about 70 amino acids to about 155 amino acids, from about 70 amino acids to about 150 amino acids, from about 70 amino acids to about 145 amino acids, from about 70 amino acids to about 140 amino acids, from about 70 amino acids to about 135 amino acids, from about 70 amino acids to about 130 amino acids, from about 70 amino acids to about 125 amino acids, from about 70 amino acids to about 120 amino acids, from about 70 amino acids to about 115 amino acids, from about 70 amino acids to about 110 amino acids, from about 70 amino acids to about 105 amino acids, from about 70 amino acids to about 100 amino acids, from about 70 amino acids to about 95 amino acids, from about 70 amino acids to about 90 amino acids, from about 70 amino acids to about 85 amino acids, from about 70 amino acids to about 80 amino acids, from about 80 amino acids to about 220 amino acids, from about 80 amino acids to about 215 amino acids, from about 80 amino acids to about 210 amino acids, from about 80 amino acids to about 205 amino acids, from about 80 amino acids to about 200 amino acids, from about 80 amino acids to about 195 amino acids, from about 80 amino acids to about 190 amino acids, from about 80 amino acids to about 185 amino acids, from about 80 amino acids to about 180 amino acids, from about 80 amino acids to about 175 amino acids, from about 80 amino acids to about 170 amino acids, from about 80 amino acids to about 165 amino acids, from about 80 amino acids to about 160 amino acids, from about 80 amino acids to about 155 amino acids, from about 80 amino acids to about 150 amino acids, from about 80 amino acids to about 145 amino acids, from about 80 amino acids to about 140 amino acids, from about 80 amino acids to about 135 amino acids, from about 80 amino acids to about 130 amino acids, from about 80 amino acids to about 125 amino acids, from about 80 amino acids to about 120 amino acids, from about 80 amino acids to about 115 amino acids, from about 80 amino acids to about 110 amino acids, from about 80 amino acids to about 105 amino acids, from about 80 amino acids to about 100 amino acids, from about 80 amino acids to about 95 amino acids, from about 80 amino acids to about 90 amino acids, from about 90 amino acids to about 220 amino acids, from about 90 amino acids to about 215 amino acids, from about 90 amino acids to about 210 amino acids, from about 90 amino acids to about 205 amino acids, from about 90 amino acids to about 200 amino acids, from about 90 amino acids to about 195 amino acids, from about 90 amino acids to about 190 amino acids, from about 90 amino acids to about 185 amino acids, from about 90 amino acids to about 180 amino acids, from about 90 amino acids to about 175 amino acids, from about 90 amino acids to about 170 amino acids, from about 90 amino acids to about 165 amino acids, from about 90 amino acids to about 160 amino acids, from about 90 amino acids to about 155 amino acids,About 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids,From about 115 amino acids to about 220 amino acids, from about 115 amino acids to about 215 amino acids, from about 115 amino acids to about 210 amino acids, from about 115 amino acids to about 205 amino acids, from about 115 amino acids to about 200 amino acids, from about 115 amino acids to about 195 amino acids, from about 115 amino acids to about 190 amino acids, from about 115 amino acids to about 185 amino acids, from about 115 amino acids ~about 180 amino acids, about 115 amino acids~about 175 amino acids, about 115 amino acids~about 170 amino acids, about 115 amino acids~about 165 amino acids, about 115 amino acids~about 160 amino acids, about 115 amino acids~about 155 amino acids, about 115 amino acids~about 150 amino acids, about 115 amino acids~about 145 amino acids, about 115 amino acids~about 140 amino acids, about 115 amino acids~about 135 amino acids, about 115 amino acids~about 130 amino acids, about 115 amino acids~about 125 amino acids, about 115 amino acids~about 120 amino acids, about 120 amino acids~about 220 amino acids, about 120 amino acids~about 215 amino acids, about 120 amino acids~about 210 amino acids, about 120 amino acids~about 205 amino acids, about 120 amino acids~about 200 amino acids, about 120 amino acids~about 195 amino acids, about 120 amino acids~about 190 amino acids, about 120 amino acids~about 185 amino acids, about 120 amino acids~about 180 amino acids, about 120 amino acids~about 175 amino acids, about 120 amino acids~about 170 amino acids, about 120 amino acids~about 165 amino acids, about 120 amino acids~about 160 amino acids, about 120 amino acids~about 155 amino acids, about 120 amino acids~about 150 amino acids, about 120 amino acids~about 145 amino acids, about 120 amino acids~about 140 amino acids, about 120 amino acids~about 135 amino acids, about 120 amino acids~about 130 amino acids, about 120 amino acids~about 125 amino acids, about 125 amino acids~about 220 amino acids, about 125 amino acids~about 215 amino acids, about 125 amino acids~about 210 amino acids, about 125 amino acids~about 205 amino acids, about 125 amino acids~about 200 amino acids, about 125 amino acids~about 195 amino acids, about 125 amino acids~about 190 amino acids, about 125 amino acids~about 185 amino acids, about 125 amino acids~about 180 amino acids, about 125 amino acids~about 175 amino acids, about 125 amino acids~about 170 amino acids, about 125 amino acids~about 165 amino acids, about 125 amino acids~about 160 amino acids, about 125 amino acids~about 155 amino acids, about 125 amino acids~about 150 amino acids, about 125 amino acids~about 145 amino acids, about 125 amino acids~about 140 amino acids, about 125 amino acids~about 135 amino acids, about 125 amino acids~about 130 amino acids, about 130 amino acids~about 220 amino acids, about 130 amino acids~about 215 amino acids, about 130 amino acids~about 210 amino acids, about 130 amino acids~about 205 amino acids,From about 130 amino acids to about 200 amino acids, from about 130 amino acids to about 195 amino acids, from about 130 amino acids to about 190 amino acids, from about 130 amino acids to about 185 amino acids, from about 130 amino acids to about 180 amino acids, from about 130 amino acids to about 175 amino acids, from about 130 amino acids to about 170 amino acids, from about 130 amino acids to about 165 amino acids, from about 130 amino acids to about 160 amino acids, from about 130 amino acids to about 155 amino acids, from about 130 amino acids to about 150 amino acids, from about 130 amino acids to about 145 amino acids, from about 130 amino acids to about 140 amino acids, from about 130 amino acids to about 135 amino acids, from about 135 amino acids to about 220 amino acids, from about 135 amino acids to about 215 amino acids, from about 135 amino acids to about 210 amino acids, from about 135 amino acids to about 205 amino acids, from about 135 amino acids to about 200 amino acids, from about 135 amino acids to about 195 amino acids, from about 135 amino acids to about 190 amino acids, from about 135 amino acids to about 185 amino acids, from about 135 amino acids to about 180 amino acids, from about 135 amino acids to about 175 amino acids, from about 135 amino acids to about 170 amino acids, from about 135 amino acids to about 165 amino acids, from about 135 amino acids to about 160 amino acids, from about 135 amino acids to about 155 amino acids, from about 135 amino acids to about 150 amino acids, from about 135 amino acids to about 145 amino acids, from about 135 amino acids to about 140 amino acids, from about 140 amino acids to about 220 amino acids, from about 140 amino acids to about 215 amino acids, from about 140 amino acids to about 210 amino acids, from about 140 amino acids to about 205 amino acids, from about 140 amino acids to about 200 amino acids, from about 140 amino acids to about 195 amino acids, from about 140 amino acids to about 190 amino acids, from about 140 amino acids to about 185 amino acids, from about 140 amino acids to about 180 amino acids, from about 140 amino acids to about 175 amino acids, from about 140 amino acids to about 170 amino acids, from about 140 amino acids to about 165 amino acids, from about 140 amino acids to about 160 amino acids, from about 140 amino acids to about 155 amino acids, from about 140 amino acids to about 150 amino acids, from about 140 amino acids to about 145 amino acids, from about 145 amino acids to about 220 amino acids, from about 145 amino acids to about 215 amino acids, from about 145 amino acids to about 210 amino acids, from about 145 amino acids to about 205 amino acids, from about 145 amino acids to about 200 amino acids, from about 145 amino acids to about 195 amino acids, from about 145 amino acids to about 190 amino acids, from about 145 amino acids to about 185 amino acidsFrom about 145 amino acids to about 180 amino acids, from about 145 amino acids to about 175 amino acids, from about 145 amino acids to about 170 amino acids, from about 145 amino acids to about 165 amino acids, from about 145 amino acids to about 160 amino acids, from about 145 amino acids to about 155 amino acids, from about 145 amino acids to about 150 amino acids, from about 150 amino acids to about 220 amino acids, from about 150 amino acids to about 215 amino acids, from about 150 amino acids to about 210 amino acids, from about 150 amino acids to about 205 amino acids, from about 150 amino acids to about 200 amino acids, from about 150 amino acids to about 195 amino acids, from about 150 amino acids to about 190 amino acids, from about 150 amino acids to about 185 amino acids, from about 150 amino acids to about 180 amino acids, from about 150 amino acids to about 175 amino acids, from about 150 amino acids to about 170 amino acids, from about 150 amino acids to about 165 amino acids, from about 150 amino acids to about 160 amino acids, from about 150 amino acids to about 155 amino acids, from about 155 amino acids to about 220 amino acids, from about 155 amino acids to about 215 amino acids, from about 155 amino acids to about 210 amino acids, from about 155 amino acids to about 205 amino acids, from about 155 amino acids to about 200 amino acids, from about 155 amino acids to about 195 amino acids, from about 155 amino acids to about 190 amino acids, from about 155 amino acids to about 185 amino acids, from about 155 amino acids to about 180 amino acids, from about 155 amino acids to about 175 amino acids, from about 155 amino acids to about 170 amino acids, from about 155 amino acids to about 165 amino acids, from about 155 amino acids to about 160 amino acids, from about 160 amino acids to about 220 amino acids, from about 160 amino acids to about 215 amino acids, from about 160 amino acids to about 210 amino acids, from about 160 amino acids to about 205 amino acids, from about 160 amino acids to about 200 amino acids, from about 160 amino acids to about 195 amino acids, from about 160 amino acids to about 190 amino acids, from about 160 amino acids to about 185 amino acids, from about 160 amino acids to about 180 amino acids, from about 160 amino acids to about 175 amino acids, from about 160 amino acids to about 170 amino acids, from about 160 amino acids to about 165 amino acids, from about 165 amino acids to about 220 amino acids, from about 165 amino acids to about 215 amino acids, from about 165 amino acids to about 210 amino acids, from about 165 amino acids to about 205 amino acids, from about 165 amino acids to about 200 amino acids, from about 165 amino acids to about 195 amino acids, from about 165 amino acids to about 190 amino acids, from about 165 amino acids to about 185 amino acids, from about 165 amino acids to about 180 amino acids,from about 165 amino acids to about 175 amino acids, from about 165 amino acids to about 170 amino acids, from about 170 amino acids to about 220 amino acids, from about 170 amino acids to about 215 amino acids, from about 170 amino acids to about 210 amino acids, from about 170 amino acids to about 205 amino acids, from about 170 amino acids to about 200 amino acids, from about 170 amino acids to about 195 amino acids, from about 170 amino acids to about 190 amino acids, from about 170 amino acids to about 185 amino acids, from about 170 amino acids to about 180 amino acids, from about 170 amino acids to about 175 amino acids, from about 175 amino acids to about 220 amino acids, from about 175 amino acids to about 215 amino acids, from about 175 amino acids to about 210 amino acids, from about 175 amino acids to about 205 amino acids, from about 175 amino acids to about 200 amino acids, from about 175 amino acids to about 195 amino acids, from about 175 amino acids to about 190 amino acids, from about 175 amino acids to about 185 amino acids, from about 175 amino acids to about 180 amino acids, from about 180 amino acids to about 220 amino acids, from about 180 amino acids to about 215 amino acids, from about 180 amino acids to about 210 amino acids, from about 180 amino acids to about 205 amino acids, from about 180 amino acids to about 200 amino acids, from about 180 amino acids to about 195 amino acids, from about 180 amino acids to about 190 amino acids, from about 180 amino acids to about 185 amino acids, from about 185 amino acids to about 220 amino acids, from about 185 amino acids to about 215 amino acids, from about 185 amino acids to about 210 amino acids, from about 185 amino acids to about 205 amino acids, from about 185 amino acids to about 200 amino acids, from about 185 amino acids to about 195 amino acids, from about 185 amino acids to about 190 amino acids, from about 190 amino acids to about 220 amino acids, from about 190 amino acids to about 215 amino acids, from about 190 amino acids to about 210 amino acids, from about 190 amino acids to about 205 amino acids, from about 190 amino acids to about 200 amino acids, from about 190 amino acids to about 195 amino acids, from about 195 amino acids to about 220 amino acids, from about 195 amino acids to about 215 amino acids, from about 195 amino acids to about 210 amino acids, from about 195 amino acids to about 205 amino acids, from about 195 amino acids to about 200 amino acids, from about 200 amino acids to about 220 amino acids, from about 200 amino acids to about 215 amino acids, from about 200 amino acids to about 210 amino acids, from about 200 amino acids to about 205 amino acids, from about 205 amino acids to about 220 amino acids, from about 205 amino acids to about 215 amino acids, from about 205 amino acids to about 210 amino acids, from about 210 amino acids to about 220 amino acids,It may have a full length of about 210 amino acids to about 215 amino acids, or about 215 amino acids to about 220 amino acids.

[0058] Soluble tissue factor domain In some examples of any of the methods, compositions, or kits described herein, the linker domain can be a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain can be a wild-type tissue factor polypeptide lacking a signal sequence, a transmembrane domain, and an intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor variant, where the wild-type tissue factor polypeptide lacks a signal sequence, a transmembrane domain, and an intracellular domain and is further modified with selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, soluble mouse tissue factor domains, soluble rat tissue factor domains, and variant soluble tissue factor domains are shown below. TIFF0007713393000026.tif72139TIFF0007713393000027.tif224138TIFF0007713393000028.tif18138

[0059] In some embodiments, the soluble tissue factor domain can comprise a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 5, 7, 8, 9, or 10. In some embodiments, the soluble tissue factor domain can comprise the sequence of SEQ ID NO: 5, 7, 8, 9, or 10, with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its N-terminus and / or 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its C-terminus.

[0060] As will be appreciated by those skilled in the art, while mutations in amino acids that are conserved among different mammalian species are likely to decrease the activity and / or structural stability of the protein, mutations in amino acids that are not conserved among different mammalian species are less likely to decrease the activity and / or structural stability of the protein.

[0061] In some instances, the soluble tissue factor domain cannot bind to factor VIIa. In some instances, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments, the single-chain chimeric polypeptide does not stimulate clotting in a mammal.

[0062] In some instances, the soluble tissue domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain.

[0063] In some examples, the soluble tissue factor domain does not include one or more (e.g., 2, 3, 4, 5, 6, or 7) of lysine at the amino acid position corresponding to amino acid 20 of the mature wild-type human tissue factor protein, isoleucine at the amino acid position corresponding to amino acid 22 of the mature wild-type human tissue factor protein, tryptophan at the amino acid position corresponding to amino acid 45 of the mature wild-type human tissue factor protein, aspartic acid at the amino acid position corresponding to amino acid 58 of the mature wild-type human tissue factor protein, tyrosine at the amino acid position corresponding to amino acid 94 of the mature wild-type human tissue factor protein, arginine at the amino acid position corresponding to amino acid 135 of the mature wild-type human tissue factor protein, and phenylalanine at the amino acid position corresponding to amino acid 140 of the mature wild-type human tissue factor protein. In some embodiments, the variant soluble tissue factor has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0064] In some examples, the soluble tissue factor domain can be encoded by a nucleic acid that includes 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: 6.

[0065] In some embodiments, the soluble tissue factor domain is from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 160 amino acids, from about 20 amino acids to about 155 amino acids, from about 20 amino acids to about 150 amino acids, from about 20 amino acids to about 145 amino acids, from about 20 amino acids to about 140 amino acids, from about 20 amino acids to about 135 amino acids, from about 20 amino acids to about 130 amino acids, from about 20 amino acids to about 125 amino acids, from about 20 amino acids to about 120 amino acids, from about 20 amino acids to about 115 amino acids, from about 20 amino acids to about 110 amino acids, from about 20 amino acids to about 105 amino acids, from about 20 amino acids to about 100 amino acids, from about 20 amino acids to about 95 amino acids, from about 20 amino acids to about 90 amino acids, from about 20 amino acids to about 85 amino acids, from about 20 amino acids to about 80 amino acids, from about 20 amino acids to about 75 amino acids, from about 20 amino acids to about 70 amino acids, from about 20 amino acids to about 60 amino acids, from about 20 amino acids to about 50 amino acids, from about 20 amino acids to about 40 amino acids, from about 20 amino acids to about 30 amino acids, from about 30 amino acids to about 220 amino acids, from about 30 amino acids to about 215 amino acids, from about 30 amino acids to about 210 amino acids, from about 30 amino acids to about 205 amino acids, from about 30 amino acids to about 200 amino acids, from about 30 amino acids to about 195 amino acids, from about 30 amino acids to about 190 amino acids, from about 30 amino acids to about 185 amino acids, from about 30 amino acids to about 180 amino acids, from about 30 amino acids to about 175 amino acids, from about 30 amino acids to about 170 amino acids, from about 30 amino acids to about 165 amino acids, from about 30 amino acids to about 160 amino acids, from about 30 amino acids to about 155 amino acids, from about 30 amino acids to about 150 amino acids, from about 30 amino acids to about 145 amino acids, from about 30 amino acids to about 140 amino acids, from about 30 amino acids to about 135 amino acids, from about 30 amino acids to about 130 amino acids, from about 30 amino acids to about 125 amino acids, from about 30 amino acids to about 120 amino acids, from about 30 amino acids to about 115 amino acids, from about 30 amino acids to about 110 amino acidsFrom about 30 amino acids to about 105 amino acids, from about 30 amino acids to about 100 amino acids, from about 30 amino acids to about 95 amino acids, from about 30 amino acids to about 90 amino acids, from about 30 amino acids to about 85 amino acids, from about 30 amino acids to about 80 amino acids, from about 30 amino acids to about 75 amino acids, from about 30 amino acids to about 70 amino acids, from about 30 amino acids to about 60 amino acids, from about 30 amino acids to about 50 amino acids, from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 220 amino acids, from about 40 amino acids to about 215 amino acids, from about 40 amino acids to about 210 amino acids, from about 40 amino acids to about 205 amino acids, from about 40 amino acids to about 200 amino acids, from about 40 amino acids to about 195 amino acids, from about 40 amino acids to about 190 amino acids, from about 40 amino acids to about 185 amino acids, from about 40 amino acids to about 180 amino acids, from about 40 amino acids to about 175 amino acids, from about 40 amino acids to about 170 amino acids, from about 40 amino acids to about 165 amino acids, from about 40 amino acids to about 160 amino acids, from about 40 amino acids to about 155 amino acids, from about 40 amino acids to about 150 amino acids, from about 40 amino acids to about 145 amino acids, from about 40 amino acids to about 140 amino acids, from about 40 amino acids to about 135 amino acids, from about 40 amino acids to about 130 amino acids, from about 40 amino acids to about 125 amino acids, from about 40 amino acids to about 120 amino acids, from about 40 amino acids to about 115 amino acids, from about 40 amino acids to about 110 amino acids, from about 40 amino acids to about 105 amino acids, from about 40 amino acids to about 100 amino acids, from about 40 amino acids to about 95 amino acids, from about 40 amino acids to about 90 amino acids, from about 40 amino acids to about 85 amino acids, from about 40 amino acids to about 80 amino acids, from about 40 amino acids to about 75 amino acids, from about 40 amino acids to about 70 amino acids, from about 40 amino acids to about 60 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 220 amino acids, from about 50 amino acids to about 215 amino acids, from about 50 amino acids to about 210 amino acids, from about 50 amino acids to about 205 amino acids, from about 50 amino acids to about 200 amino acids, from about 50 amino acids to about 195 amino acids, from about 50 amino acids to about 190 amino acids, from about 50 amino acids to about 185 amino acids, from about 50 amino acids to about 180 amino acids, from about 50 amino acids to about 175 amino acids, from about 50 amino acids to about 170 amino acids, from about 50 amino acids to about 165 amino acids, from about 50 amino acids to about 160 amino acids, from about 50 amino acids to about 155 amino acids, from about 50 amino acids to about 150 amino acidsfrom about 50 amino acids to about 145 amino acids, from about 50 amino acids to about 140 amino acids, from about 50 amino acids to about 135 amino acids, from about 50 amino acids to about 130 amino acids, from about 50 amino acids to about 125 amino acids, from about 50 amino acids to about 120 amino acids, from about 50 amino acids to about 115 amino acids, from about 50 amino acids to about 110 amino acids, from about 50 amino acids to about 105 amino acids, from about 50 amino acids to about 100 amino acids, from about 50 amino acids to about 95 amino acids, from about 50 amino acids to about 90 amino acids, from about 50 amino acids to about 85 amino acids, from about 50 amino acids to about 80 amino acids, from about 50 amino acids to about 75 amino acids, from about 50 amino acids to about 70 amino acids, from about 50 amino acids to about 60 amino acids, from about 60 amino acids to about 220 amino acids, from about 60 amino acids to about 215 amino acids, from about 60 amino acids to about 210 amino acids, from about 60 amino acids to about 205 amino acids, from about 60 amino acids to about 200 amino acids, from about 60 amino acids to about 195 amino acids, from about 60 amino acids to about 190 amino acids, from about 60 amino acids to about 185 amino acids, from about 60 amino acids to about 180 amino acids, from about 60 amino acids to about 175 amino acids, from about 60 amino acids to about 170 amino acids, from about 60 amino acids to about 165 amino acids, from about 60 amino acids to about 160 amino acids, from about 60 amino acids to about 155 amino acids, from about 60 amino acids to about 150 amino acids, from about 60 amino acids to about 145 amino acids, from about 60 amino acids to about 140 amino acids, from about 60 amino acids to about 135 amino acids, from about 60 amino acids to about 130 amino acids, from about 60 amino acids to about 125 amino acids, from about 60 amino acids to about 120 amino acids, from about 60 amino acids to about 115 amino acids, from about 60 amino acids to about 110 amino acids, from about 60 amino acids to about 105 amino acids, from about 60 amino acids to about 100 amino acids, from about 60 amino acids to about 95 amino acids, from about 60 amino acids to about 90 amino acids, from about 60 amino acids to about 85 amino acids, from about 60 amino acids to about 80 amino acids, from about 60 amino acids to about 75 amino acids, from about 60 amino acids to about 70 amino acids, from about 70 amino acids to about 220 amino acids, from about 70 amino acids to about 215 amino acids, from about 70 amino acids to about 210 amino acids, from about 70 amino acids to about 205 amino acids, from about 70 amino acids to about 200 amino acids, from about 70 amino acids to about 195 amino acids, from about 70 amino acids to about 190 amino acids, from about 70 amino acids to about 185 amino acids, from about 70 amino acids to about 180 amino acids, from about 70 amino acids to about 175 amino acids, from about 70 amino acids to about 170 amino acidsFrom about 70 amino acids to about 165 amino acids, from about 70 amino acids to about 160 amino acids, from about 70 amino acids to about 155 amino acids, from about 70 amino acids to about 150 amino acids, from about 70 amino acids to about 145 amino acids, from about 70 amino acids to about 140 amino acids, from about 70 amino acids to about 135 amino acids, from about 70 amino acids to about 130 amino acids, from about 70 amino acids to about 125 amino acids, from about 70 amino acids to about 120 amino acids, from about 70 amino acids to about 115 amino acids, from about 70 amino acids to about 110 amino acids, from about 70 amino acids to about 105 amino acids, from about 70 amino acids to about 100 amino acids, from about 70 amino acids to about 95 amino acids, from about 70 amino acids to about 90 amino acids, from about 70 amino acids to about 85 amino acids, from about 70 amino acids to about 80 amino acids, from about 80 amino acids to about 220 amino acids, from about 80 amino acids to about 215 amino acids, from about 80 amino acids to about 210 amino acids, from about 80 amino acids to about 205 amino acids, from about 80 amino acids to about 200 amino acids, from about 80 amino acids to about 195 amino acids, from about 80 amino acids to about 190 amino acids, from about 80 amino acids to about 185 amino acids, from about 80 amino acids to about 180 amino acids, from about 80 amino acids to about 175 amino acids, from about 80 amino acids to about 170 amino acids, from about 80 amino acids to about 165 amino acids, from about 80 amino acids to about 160 amino acids, from about 80 amino acids to about 155 amino acids, from about 80 amino acids to about 150 amino acids, from about 80 amino acids to about 145 amino acids, from about 80 amino acids to about 140 amino acids, from about 80 amino acids to about 135 amino acids, from about 80 amino acids to about 130 amino acids, from about 80 amino acids to about 125 amino acids, from about 80 amino acids to about 120 amino acids, from about 80 amino acids to about 115 amino acids, from about 80 amino acids to about 110 amino acids, from about 80 amino acids to about 105 amino acids, from about 80 amino acids to about 100 amino acids, from about 80 amino acids to about 95 amino acids, from about 80 amino acids to about 90 amino acids, from about 90 amino acids to about 220 amino acids, from about 90 amino acids to about 215 amino acids, from about 90 amino acids to about 210 amino acids, from about 90 amino acids to about 205 amino acids, from about 90 amino acids to about 200 amino acids, from about 90 amino acids to about 195 amino acids, from about 90 amino acids to about 190 amino acids, from about 90 amino acids to about 185 amino acids, from about 90 amino acids to about 180 amino acids, from about 90 amino acids to about 175 amino acids, from about 90 amino acids to about 170 amino acids, from about 90 amino acids to about 165 amino acids, from about 90 amino acids to about 160 amino acids, from about 90 amino acids to about 155 amino acids,About 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids,From about 115 amino acids to about 220 amino acids, from about 115 amino acids to about 215 amino acids, from about 115 amino acids to about 210 amino acids, from about 115 amino acids to about 205 amino acids, from about 115 amino acids to about 200 amino acids, from about 115 amino acids to about 195 amino acids, from about 115 amino acids to about 190 amino acids, from about 115 amino acids to about 185 amino acids, about 115 a, Amino acids ~ about 180 amino acids, about 115 amino acids ~ about 175 amino acids, about 115 amino acids ~ about 170 amino acids, about 115 amino acids ~ about 165 amino acids, about 115 amino acids ~ about 160 amino acids, about 115 amino acids ~ about 155 amino acids, about 115 amino acids ~ about 150 amino acids, about 115 amino acids ~ about 145 amino acids, about 115 amino acids ~ about 140 amino acids, about 115 amino acids ~ about 135 amino acids, about 115 amino acids ~ about 130 amino acids, about 115 amino acids ~ about 125 amino acids, about 115 amino acids ~ about 120 amino acids, about 120 amino acids ~ about 220 amino acids, about 120 amino acids ~ about 215 amino acids, about 120 amino acids ~ about 210 amino acids, about 120 amino acids ~ about 205 amino acids, about 120 amino acids ~ about 200 amino acids, about 120 amino acids ~ about 195 amino acids, about 120 amino acids ~ about 190 amino acids, about 120 amino acids ~ about 185 amino acids, about 120 amino acids ~ about 180 amino acids, about 120 amino acids ~ about 175 amino acids, about 120 amino acids ~ about 170 amino acids, about 120 amino acids ~ about 165 amino acids, about 120 amino acids ~ about 160 amino acids, about 120 amino acids ~ about 155 amino acids, about 120 amino acids ~ about 150 amino acids, about 120 amino acids ~ about 145 amino acids, about 120 amino acids ~ about 140 amino acids, about 120 amino acids ~ about 135 amino acids, about 120 amino acids ~ about 130 amino acids, about 120 amino acids ~ about 125 amino acids, about 125 amino acids ~ about 220 amino acids, about 125 amino acids ~ about 215 amino acids, about 125 amino acids ~ about 210 amino acids, about 125 amino acids ~ about 205 amino acids, about 125 amino acids ~ about 200 amino acids, about 125 amino acids ~ about 195 amino acids, about 125 amino acids ~ about 190 amino acids, about 125 amino acids ~ about 185 amino acids, about 125 amino acids ~ about 180 amino acids, about 125 amino acids ~ about 175 amino acids, about 125 amino acids ~ about 170 amino acids, about 125 amino acids ~ about 165 amino acids, about 125 amino acids ~ about 160 amino acids, about 125 amino acids ~ about 155 amino acids, about 125 amino acids ~ about 150 amino acids, about 125 amino acids ~ about 145 amino acids, about 125 amino acids ~ about 140 amino acids, about 125 amino acids ~ about 135 amino acids, about 125 amino acids ~ about 130 amino acids, about 130 amino acids ~ about 220 amino acids, about 130 amino acids ~ about 215 amino acids, about 130 amino acids ~ about 210 amino acidsfrom about 130 amino acids to about 205 amino 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Claims

1. A method for promoting the activation and proliferation of natural killer cells (NK cells), comprising: (a) contacting natural killer cells with an effective amount of a first multi-chain chimeric polypeptide in a liquid culture medium for 15 minutes to 1 day under conditions that allow differentiation of the natural killer cells; and (b) contacting the natural killer cells with an effective amount of (i) a second multi-chain chimeric polypeptide and (ii) an IgG1 antibody construct in a liquid culture medium for 1 day to 30 days under conditions that allow activation and proliferation of the natural killer cells; wherein the first multi-chain chimeric polypeptide comprises a first target-binding domain, a soluble tissue factor domain comprising a sequence at least 90% identical to SEQ ID NO: 5, a first domain of an affinity domain pair comprising a sequence at least 90% identical to SEQ ID NO: 26, to form a first chimeric polypeptide, and a second domain of the affinity domain pair comprising a sequence at least 90% identical to SEQ ID NO: 24, a second target-binding domain to form a second chimeric polypeptide; wherein the first chimeric polypeptide and the second chimeric polypeptide within the first multi-chain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the first multi-chain chimeric polypeptide; wherein (A) the first target-binding domain within the first multi-chain chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 20, and the second target-binding domain within the first multi-chain chimeric polypeptide comprises a first sequence at least 90% identical to SEQ ID NO: 14 and a second sequence at least 90% identical to SEQ ID NO: 16, or (B) the first target-binding domain within the first multi-chain chimeric polypeptide comprises a first sequence at least 90% identical to SEQ ID NO: 14 and a second sequence at least 90% identical to SEQ ID NO: 16, and the second target-binding domain within the first multi-chain chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO: 20; the second multi-chain chimeric polypeptide comprises a first target-binding domain, a soluble tissue factor domain comprising a sequence at least 90% identical to SEQ ID NO: 5, a first domain of an affinity domain pair comprising a sequence at least 90% identical to SEQ ID NO: 26, to form a first chimeric polypeptide, A second domain of an affinity domain pair, comprising a sequence that is at least 90% identical to SEQ ID NO: 24, and a second target binding domain comprising a second chimeric polypeptide, comprising the first chimeric polypeptide and the second chimeric polypeptide within the second multichimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the second multichimeric polypeptide, either (A) the first target binding domain within the second multichimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 23 and the second target binding domain within the second multichimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 22, or (B) the first target binding domain within the second multichimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 22 and the second target binding domain within the second multichimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 23, and the IgG1 antibody construct comprises at least one antigen-binding domain that specifically binds to the soluble tissue factor domain, the method. **Claim 2** The method according to claim 1, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide of the first multichimeric polypeptide. **Claim 3** The method according to claim 1, wherein the first chimeric polypeptide within the first multichimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide. **Claim 4** The method according to any one of claims 1 to 3, wherein the soluble tissue factor domain and the first domain of the affinity domain pair within the first chimeric polypeptide of the first multichimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide. **Claim 5** The method according to any one of claims 1 to 3, wherein the first chimeric polypeptide within the first multichimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair within the first chimeric polypeptide. **Claim 6** The method according to any one of claims 1 to 5, wherein the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide within the first multichain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide.

7. The method according to any one of claims 1 to 5, wherein the second chimeric polypeptide within the first multichain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide.

8. The method according to any one of claims 1 to 7, wherein the first multichain chimeric polypeptide and the second multichain chimeric polypeptide do not stimulate coagulation in mammals.

9. The method according to any one of claims 1 to 8, wherein the first target binding domain within the first multichain chimeric polypeptide specifically binds to the IL-12 receptor, and the second target binding domain within the first multichain chimeric polypeptide specifically binds to the IL-18 receptor.

10. The method according to any one of claims 1 to 8, wherein the first target binding domain within the first multichain chimeric polypeptide specifically binds to the IL-18 receptor, and the second target binding domain within the first multichain chimeric polypeptide specifically binds to the IL-12 receptor.

11. The first target binding domain within the first multichain chimeric polypeptide comprises soluble human IL-18, and the second target binding domain within the first multichain chimeric polypeptide comprises soluble human IL-12. The method according to claim 10.

12. The first chimeric polypeptide within the first multichain chimeric polypeptide comprises a sequence that is at least 90% identical to, and the second chimeric polypeptide comprises a sequence that is at least 90% identical to. The method according to claim 1.

13. The method according to any one of claims 1 to 12, wherein the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide within the second multichain chimeric polypeptide are directly adjacent to each other within the first chimeric polypeptide.

14. The method according to any one of claims 1 to 12, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

15. The method according to any one of claims 1 to 14, wherein the soluble tissue factor domain and the first domain of the affinity domain pair are directly adjacent to each other within the first chimeric polypeptide of the second multichain chimeric polypeptide.

16. The method according to any one of claims 1 to 14, wherein the first chimeric polypeptide within the second multichain chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair within the first chimeric polypeptide.

17. The method according to any one of claims 1 to 16, wherein the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide of the second multichain chimeric polypeptide are directly adjacent to each other within the second chimeric polypeptide.

18. The method according to any one of claims 1 to 16, wherein the second chimeric polypeptide within the second multichain chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain within the second chimeric polypeptide.

19. The method according to any one of claims 1 to 18, wherein the first target binding domain and the second target binding domain within the second multichain chimeric polypeptide are each independently soluble IL-7 or soluble IL-21.

20. The method according to any one of claims 1 to 19, wherein the first target binding domain within the second multichain chimeric polypeptide specifically binds to the IL-21 receptor, and the second target binding domain within the second multichain chimeric polypeptide specifically binds to the IL-7 receptor.

21. The method according to any one of claims 1 to 19, wherein the first target binding domain within the second multichain chimeric polypeptide specifically binds to the IL-7 receptor, and the second target binding domain within the second multichain chimeric polypeptide specifically binds to the IL-21 receptor.

22. The first chimeric polypeptide within the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to and the second chimeric polypeptide within the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to The method according to claim 1.

23. The first chimeric polypeptide within the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to and the second chimeric polypeptide within the second multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to The method according to claim 1.

24. The method according to any one of claims 1 to 23, wherein the NK cells have been previously genetically modified to express a chimeric antigen receptor or a recombinant T cell receptor.

25. The method according to any one of claims 1 to 24, further comprising isolating the NK cells after step (b).

26. (a) A first multi-chain chimeric polypeptide comprising a first target binding domain, a soluble tissue factor domain comprising a sequence that is at least 90% identical to SEQ ID NO: 5, a first domain of an affinity domain pair comprising a sequence that is at least 90% identical to SEQ ID NO: 26 and a first chimeric polypeptide, and a second domain of the affinity domain pair comprising a sequence that is at least 90% identical to SEQ ID NO: 24 and a second target binding domain and a second chimeric polypeptide wherein the first chimeric polypeptide and the second chimeric polypeptide within the first multi-chain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the first multi-chain chimeric polypeptide, (A) The first target binding domain within the first multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 20, and the second target binding domain within the first multi-chain chimeric polypeptide comprises a first sequence that is at least 90% identical to SEQ ID NO: 14 and a second sequence that is at least 90% identical to SEQ ID NO: 16, or (B) The first target binding domain within the first multi-chain chimeric polypeptide comprises a first sequence that is at least 90% identical to SEQ ID NO: 14 and a second sequence that is at least 90% identical to SEQ ID NO: 16, and the second target binding domain within the first multi-chain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:

20. ​ said first multichain chimeric polypeptide, and (b) a second multichain chimeric polypeptide, a first target binding domain, a soluble tissue factor domain comprising a sequence that is at least 90% identical to SEQ ID NO: 5, a first domain of an affinity domain pair comprising a sequence that is at least 90% identical to SEQ ID NO: 26, and a first chimeric polypeptide, and a second domain of an affinity domain pair comprising a sequence that is at least 90% identical to SEQ ID NO: 24, a second target binding domain a second chimeric polypeptide comprising, wherein the first chimeric polypeptide and the second chimeric polypeptide within the second multichain chimeric polypeptide associate by binding of the first domain and the second domain of the affinity domain pair within the second multichain chimeric polypeptide, (A) the first target binding domain within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 23, and the second target binding domain within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 22, or (B) the first target binding domain within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 22, and the second target binding domain within the second multichain chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 23, the second multichain chimeric polypeptide, and (c) an IgG1 antibody comprising at least one antigen binding domain that specifically binds to the soluble tissue factor domain A kit comprising.

Images