Multichain chimeric polypeptides and uses thereof

Abstract

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

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 62 / 864,996, filed June 21, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biotechnology, and more specifically, to the use of a combination of two multi-chain chimeric polypeptides, each having a linker domain located between two target-binding domains. Background Technology

[0004] Adoptive immunotherapy or cell therapy requires culturing immune cells obtained from a subject in vivo (and optionally genetically modifying the immune cells to express T cells or chimeric antigen receptors) before administering them back into the subject. A sufficient number of immune cells is required to provide therapeutic efficacy in the subject. In many cases, culturing immune cells obtained from the subject for three weeks or more is necessary to obtain a therapeutically effective number of immune cells. Furthermore, many methods for culturing immune cells obtained from the subject in vitro require a layer of feeder cells, which necessitates subsequent purification or isolation of the immune cells before re-administration into the subject. Summary of the Invention

[0005] This invention is based on the discovery that a specific two-step method for contacting NK cells leads to significant expansion of NK cells in vitro, and that the expanded NK cells are highly activated and exhibit long-term persistence and high cytotoxicity to diseased cells in the recipient host.

[0006] In one aspect, this article provides a method for promoting the activation and proliferation of natural killer cells, the method comprising: (a) contacting the natural killer cells in a liquid culture medium containing an effective amount of a first multi-chain chimeric polypeptide under conditions allowing for the differentiation of natural killer cells, for a first time period; and (b) contacting the natural killer cells in a liquid culture medium containing an effective amount of (i) a second multi-chain chimeric polypeptide and (ii) an IgG1 antibody construct under conditions allowing for the activation and proliferation of the natural killer cells, for a second time period, wherein the first multi-chain chimeric polypeptide comprises: a first chimeric polypeptide comprising a first domain of a first target-binding domain, a soluble tissue factor domain, and a pair of affinity domains; and a second chimeric polypeptide comprising a second domain of a pair of affinity domains and a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide in the first multi-chain chimeric polypeptide are connected by the first domain of the affinity domain pair in the first multi-chain chimeric polypeptide and the second chimeric polypeptide. The second multi-chain chimeric polypeptide associates with the first target-binding domain and the second target-binding domain of the first multi-chain chimeric polypeptide, and each independently and specifically binds to the IL-18 receptor or the IL-12 receptor; and wherein the second multi-chain chimeric polypeptide comprises: a first chimeric polypeptide comprising a first target-binding domain, a linker domain, and a first domain of a pair of affinity domains; and a second chimeric polypeptide comprising a second domain of a pair of affinity domains and a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the affinity domain pair in the second multi-chain chimeric polypeptide; the first target-binding domain and the second target-binding domain of the second multi-chain chimeric polypeptide independently and specifically bind to the IL-7 receptor or the IL-21 receptor, and the IgG1 antibody construct comprises at least one antigen-binding domain specifically bound to the linker domain.

[0007] In some embodiments, the first target-binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide of the first multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide of the first multi-chain chimeric polypeptide further includes a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments, the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide of the first multi-chain chimeric polypeptide are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide of the first multi-chain chimeric polypeptide further includes a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide. In some embodiments, the second domain of the affinity domain pair and the second target-binding domain in the second chimeric polypeptide of the first multi-chain chimeric polypeptide are directly adjacent to each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide of the first multi-chain chimeric polypeptide further includes a linker sequence between the second domain of the affinity domain pair and the second target-binding domain in 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 having at least 80% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 90% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 90% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 90% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 95% identity with 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 the following: lysine at amino acid position 20 corresponding to amino acid position 2 ... In some embodiments, the soluble human tissue factor domain does not include any of the following: lysine at amino acid position 20 corresponding to amino acid position 22 corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at amino acid position 45 corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at amino acid position 94 corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at amino acid position 135 corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X into 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 in the first multi-chain chimeric polypeptide further include 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 in the first multi-chain chimeric polypeptide is a sushi domain from the α chain (IL15Rα) of the human IL-15 receptor and soluble IL-15. In some embodiments, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, the soluble IL-15 comprises a sequence having 80% identity with SEQ ID NO:26.In some embodiments, the soluble IL-15 comprises a sequence having 90% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 comprises a sequence having 95% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 comprises SEQ ID NO:26. In some embodiments, the sushi domain of the IL15Rα comprises a sushi domain derived from human IL15Rα. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 80% identity with SEQ ID NO:24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 90% identity with SEQ ID NO:24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 95% identity with 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 mature, full-length IL15Rα.

[0008] In some embodiments, the affinity domain pairs in the first multi-chain chimeric polypeptide are selected from the group consisting of: barnase and barnstar, PKA and AKAP, an adaptor / docking tag module based on a mutant ribonuclease I fragment, and a SNARE module based on the interaction of protein synaptic fusion proteins, synaptic binding proteins, synaptic vesicle proteins, and SNAP25. In some embodiments, 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. In some embodiments, 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. 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 having at least 80% identity with SEQ ID NO:20. In some embodiments, the soluble human IL-18 comprises a sequence having at least 90% identity with SEQ ID NO:20. In some embodiments, the soluble human IL-18 comprises a sequence having at least 95% identity with 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 a soluble IL-12β (p40) sequence and a 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 having at least 80% identity with SEQ ID NO:14. In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence having at least 90% identity with SEQ ID NO:14.In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:16. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence having at least 90% identity with SEQ ID NO:16. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence having at least 95% identity with 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 in the first multi-chain chimeric polypeptide comprises […].

[0009] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGY FLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAG The sequence EPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 63) has at least 80% identity. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide comprises a sequence having at least 95% identity with SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide comprises SEQ ID NO: 63. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide comprises

[0010] MKWVTFISLLFLFSSAYSYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGTTNTVAAYNLTWKSTNFKTILEWKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAG NVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:64)。 In some embodiments, the second part of the article is included

[0011] (SEQ ID NO:65) has at least 80% identity with the sequence. In some embodiments, the second chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO:65. In some embodiments, the second chimeric polypeptide comprises a sequence having at least 95% identity with SEQ ID NO:65. In some embodiments, the second chimeric polypeptide comprises SEQ ID NO:65. In some embodiments, the second chimeric polypeptide comprises...

[0012] (SEQ ID NO: 66). In some embodiments, the first target-binding domain and the linker domain of the first chimeric peptide in the second multi-chain chimeric peptide are directly adjacent to each other in the first chimeric peptide. In some embodiments, the first chimeric peptide in the second multi-chain chimeric peptide further includes a linker sequence between the first target-binding domain and the linker domain in the first chimeric peptide. In some embodiments, the linker domain and the first domain of the affinity domain pair are directly adjacent to each other in the first chimeric peptide in the second multi-chain chimeric peptide. In some embodiments, the first chimeric peptide in the second multi-chain chimeric peptide further includes a linker sequence between the linker domain and the first domain of the affinity domain pair in the first chimeric peptide. In some embodiments, the second domain of the affinity domain pair and the second target-binding domain of the second chimeric peptide in the second multi-chain chimeric peptide are directly adjacent to each other in the second chimeric peptide.In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide further comprises a linker sequence between the second domain and the second target-binding domain of the affinity pair in 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 having at least 80% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 90% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 90% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 90% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain comprises a sequence having at least 95% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain comprises SEQ ID NO:10.

[0013] In some embodiments, the soluble human tissue factor domain does not include one or more of the following: lysine at amino acid position 20 corresponding to amino acid position 22 corresponding to amino acid position 22 corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at amino acid position 45 corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at amino acid position 94 corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at amino acid position 135 corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of the following: lysine at amino acid position 20 corresponding to mature wild-type human tissue factor protein; isoleucine at amino acid position 22 corresponding to mature wild-type human tissue factor protein; tryptophan at amino acid position 45 corresponding to mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 corresponding to mature wild-type human tissue factor protein; tyrosine at amino acid position 94 corresponding to mature wild-type human tissue factor protein; arginine at amino acid position 135 corresponding to mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 corresponding to mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X into factor Xa. In some embodiments, the second multi-chain chimeric polypeptide does not stimulate coagulation in mammals. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide in the second multi-chain chimeric polypeptide further include 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 in the second multi-chain chimeric polypeptide is a sushi domain from the α chain (IL15Rα) of the human IL-15 receptor and soluble IL-15. In some embodiments, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, the soluble IL-15 contains a sequence having 80% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 contains a sequence having 90% identity with SEQ ID NO:26.In some embodiments, the soluble IL-15 comprises a sequence having 95% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 comprises SEQ ID NO:26. In some embodiments, the sushi domain of the IL15Rα comprises a sushi domain derived from human IL15Rα. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 80% identity with SEQ ID NO:24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 90% identity with SEQ ID NO:24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 95% identity with 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 mature, full-length IL15Rα. In some embodiments, the affinity domain pairs in the second multi-chain chimeric polypeptide are selected from the group consisting of: barnase and barnstar, PKA and AKAP, an adaptor / docking tag module based on a mutant ribonuclease I fragment, and a SNARE module based on the interaction of protein synaptic fusion proteins, synaptic binding proteins, synaptic vesicle proteins, 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 contains 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 having at least 80% identity with SEQ ID NO:22. In some embodiments, the soluble human IL-21 comprises a sequence having at least 90% identity with SEQ ID NO:22. In some embodiments, the soluble human IL-21 comprises a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:23. In some embodiments, the soluble human IL-7 comprises a sequence having at least 90% identity with SEQ ID NO:23. In some embodiments, the soluble human IL-7 comprises a sequence having at least 95% identity with SEQ ID NO:23. In some embodiments, the soluble human IL-7 comprises the sequence of SEQ ID NO:23.

[0014] In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises and

[0015] DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQ KKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEP The sequence LYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 67) has at least 80% identity. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO: 67. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 95% identity with 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 comprises

[0016] MKWVTFISLLFLFSSAYSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNV ESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 68). In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises with

[0017] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 69) has a sequence having at least 80% identity. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 95% identity with SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 69. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises...

[0018] MKWVTFISLLFLFSSAYSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 70). In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises with

[0019] QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQK MIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTP YLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 71) has a sequence with at least 80% identity. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence with at least 90% identity to SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence with at least 95% identity to SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 71. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises

[0020] MGVKVLFALICIAVAEAQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPP KEFLERFKSLLQKMIHQHLSSRTHGSEDSSGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPL YENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRENWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 72). In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises with

[0021] DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 73) has a sequence with at least 80% identity. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence with at least 90% identity to SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence with at least 95% identity to SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO: 73. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises...

[0022] MGVKVLFALICIAVAEADCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 74). In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the adapter domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the adapter domain. In some embodiments, the first time period is from about 15 minutes to about 4 hours. In some embodiments, the first time period is from about 30 minutes to about 3.5 hours. In some embodiments, the first time period is from about 1 hour to about 3.5 hours. In some embodiments, the first time period is from about 2 hours to about 3.5 hours. In some embodiments, the liquid culture medium in step (a) and / or step (b) is a serum-containing liquid culture medium.

[0023] In some embodiments, the NK cells are at approximately 4.0 x 10⁻⁶. 7 Cells / mL to approximately 1.5 x 10⁻⁶ 8 The NK cells are present in the liquid culture medium of step (a) at a density of approximately 5.0 x 10⁶ cells / mL. In some embodiments, the NK cells are present at a density of approximately 5.0 x 10⁶ cells / mL. 7 Cells / mL to approximately 2.0 x 10⁻⁶ cells / mL 8The NK cells are present in the liquid culture medium in step (a) at a density of cells / mL. In some embodiments, the second time period is about 1 day to about 30 days. In some embodiments, the second time period is about 7 days to about 30 days. In some embodiments, the second time period is about 14 days to about 21 days. In some embodiments, the liquid culture medium in step (b) comprises the second multi-chain chimeric peptide and the IgG1 antibody construct in a molar ratio of about 0.5:1 to about 2:1. In some embodiments, the liquid culture medium in step (b) comprises the second multi-chain chimeric peptide and the IgG1 antibody construct in a molar ratio of about 0.8:1 to about 1.2:1. In some embodiments, the NK cells are maintained at a density of about 0.5 x 10⁻⁶ cells / mL in the liquid culture medium in step (b). 6 Cells / mL to approximately 2.0 x 10⁻⁶ cells / mL 6 A density of cells / mL. In some embodiments, the NK cells are maintained at approximately 1.0 x 10⁻⁶ cells / mL in the liquid culture medium in step (b). 6 Cells / mL to approximately 2.0 x 10⁻⁶ cells / mL 6A density of cells / mL. In some embodiments, the NK cells are previously obtained from a subject. In some embodiments, the NK cells are previously obtained from umbilical cord blood or induced pluripotent stem cells. In some embodiments, the method further comprises obtaining the NK cells from the 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 steps (a) and (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), compared with the expression or secretion level of one or more proteins prior to step (a), the expression or secretion level of one or more proteins on the NK cells increases, wherein the one or more proteins are 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, CD 94, 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. In some embodiments, the method further comprises administering the NK cells to a subject in need after the contact step. In some embodiments, the subject has been identified or diagnosed with 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, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, and renal dysfunction. In some embodiments, the subject has been identified or diagnosed with cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing sarcoma, osteosarcoma, B-cell neoplasm, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, and Hodgkin's lymphoma. Chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been diagnosed or identified as having an infectious disease. In some embodiments, the infectious disease is infection with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus, hepatitis C virus, human papillomavirus, and influenza virus.

[0024] This document also provides activated NK cells generated by the method described in any of the above embodiments. A pharmaceutical composition comprising the activated NK cells described in the above embodiments is also provided. A kit comprising a pharmaceutical composition containing the activated NK cells described in any of the above embodiments is also provided. In another aspect, this document provides a method for killing cancer cells, infected cells, or senescent cells in a subject in need, the method comprising administering to the subject a therapeutically effective amount of any of the activated NK cells described in the above embodiments or any of the pharmaceutical compositions described in the above embodiments. In some embodiments, the subject has been identified or diagnosed with cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed with an age-related disease or condition. In some embodiments, the age-related diseases or conditions are selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung 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 with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus and hepatitis C virus, human papillomavirus and influenza virus.

[0025] In some embodiments, this document provides a method of treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of any of the activated NK cells or activated T cells as described in any of the above embodiments, or any of the pharmaceutical compositions described in the above embodiments. In some embodiments, the subject has been identified or diagnosed with cancer. In some embodiments, the cancer is selected from the group consisting of: solid tumors, hematologic malignancies, sarcomas, osteosarcomas, glioblastoma, neuroblastoma, melanoma, rhabdomyosarcoma, Ewing's sarcoma, osteosarcoma, B-cell neoplasms, multiple myeloma, B-cell lymphoma, B-cell non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma, retinoblastoma, gastric cancer, urothelial carcinoma, lung cancer, renal cell carcinoma, gastric and esophageal cancer, pancreatic cancer, prostate cancer, breast cancer, colorectal cancer, ovarian cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, cervical cancer, liver cancer, and hepatocellular carcinoma. In some embodiments, the subject has been identified or diagnosed with an age-related disease or condition. In some embodiments, the age-related diseases or conditions are selected from the group consisting of: Alzheimer's disease, aneurysm, cystic fibrosis, pancreatic fibrosis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes, lipomatosis, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, failed kidney transplant, liver fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, hair loss, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung 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 with human immunodeficiency virus, cytomegalovirus, adenovirus, coronavirus, rhinovirus, rotavirus, smallpox, herpes simplex virus, hepatitis B virus, hepatitis A virus and hepatitis C virus, human papillomavirus and influenza virus.

[0026] On the other hand, this document provides a kit comprising: (a) a first multi-chain chimeric polypeptide, the first multi-chain chimeric polypeptide comprising: a first chimeric polypeptide including a first target-binding domain, a soluble tissue factor domain, and a first domain of a pair of affinity domains; and a second chimeric polypeptide including a second domain of a pair of affinity domains and a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide in the first multi-chain chimeric polypeptide are associated by binding of the first domain and the second domain of the affinity domain pair in the first multi-chain chimeric polypeptide, and the first target-binding domain and the second target-binding domain in the first multi-chain chimeric polypeptide each independently and specifically bind to an IL-18 receptor or an IL-12 receptor; the second multi-chain chimeric polypeptide The second multi-chain chimeric polypeptide comprises: a first chimeric polypeptide including a first target-binding domain, a linker domain, and a first domain of a pair of affinity domains; and a second chimeric polypeptide including a second domain of a pair of affinity domains and a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide in the second multi-chain chimeric polypeptide are associated by binding of the first and second domains of the affinity domain pair in the second multi-chain chimeric polypeptide; and the first target-binding domain and the second target-binding domain in the second multi-chain chimeric polypeptide each independently and specifically bind to an IL-7 receptor or an IL-21 receptor; and (c) an IgG1 antibody, the IgG1 antibody including at least one antigen-binding domain specifically bound to the linker domain. In some embodiments, the first target-binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide of the first multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide further includes a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments, the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide. In some embodiments, the second domain of the affinity domain pair and the second target-binding domain in the second chimeric polypeptide are directly adjacent to each other in the second chimeric polypeptide.In some embodiments, the second chimeric polypeptide in the first multi-chain chimeric polypeptide further includes a linker sequence between the second domain and the second target-binding domain of the affinity pair in 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 includes a sequence having at least 80% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 90% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 90% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 95% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 80% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 90% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 95% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain includes SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain does not include one or more of the following: lysine at amino acid position 20 corresponding to amino acid position 22 corresponding to amino acid position 22 corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at amino acid position 45 corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at amino acid position 94 corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at amino acid position 135 corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.In some embodiments, the soluble human tissue factor domain does not include any of the following: lysine at amino acid position 20 corresponding to amino acid position 22 corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at amino acid position 45 corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at amino acid position 94 corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at amino acid position 135 corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X into 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 in the first multi-chain chimeric polypeptide further include 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 in the first multi-chain chimeric polypeptide is a sushi domain from the α chain (IL15Rα) of the human IL-15 receptor and soluble IL-15. In some embodiments, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, the soluble IL-15 includes a sequence having 80% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 includes a sequence having 90% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 includes a sequence having 95% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 includes SEQ ID NO:26. In some embodiments, the sushi domain of the IL15Rα includes a sushi domain from human IL15Rα. In some embodiments, the sushi domain from human IL15Rα includes a sequence having 80% identity with SEQ ID NO:24. In some embodiments, the sushi domain from human IL15Rα includes a sequence having 90% identity with SEQ ID NO:24. In some embodiments, the sushi domain from human IL15Rα includes a sequence having 95% identity with SEQ ID NO:24.In some embodiments, the sushi domain from human IL15Rα includes SEQ ID NO:24. In some embodiments, the sushi domain from human IL15Rα is mature, full-length IL15Rα. In some embodiments, the affinity domain pairs in the first multi-chain chimeric polypeptide are selected from the group consisting of: barnase and barnstar, PKA and AKAP, adaptor / docking tag modules based on mutant ribonuclease I fragments, and SNARE modules based on the interaction of protein synaptic fusion proteins, synaptic binding proteins, synaptic vesicle proteins, and SNAP25. In some embodiments, 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. In some embodiments, 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. In some embodiments, the first target-binding domain of the first multi-chain chimeric polypeptide specifically binds to the IL-18 receptor, and the second target-binding domain of the first multi-chain chimeric polypeptide specifically binds to the IL-12 receptor. In some embodiments, the first target-binding domain of 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 having at least 80% identity with SEQ ID NO:20. In some embodiments, the soluble human IL-18 comprises a sequence having at least 90% identity with SEQ ID NO:20. In some embodiments, the soluble human IL-18 comprises a sequence having at least 95% identity with 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 of 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 having at least 80% identity with SEQ ID NO:14.In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence having at least 90% identity with SEQ ID NO:14. In some embodiments, the soluble human IL-12β (p40) sequence comprises a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:16. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence having at least 90% identity with SEQ ID NO:16. In some embodiments, the soluble human IL-12α (p35) sequence comprises a sequence having at least 95% identity with 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 in the first multi-chain chimeric polypeptide includes a sequence having at least 80% identity with SEQ ID NO:63. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide includes a sequence having at least 90% identity with SEQ ID NO:63. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide includes a sequence having at least 95% identity with SEQ ID NO:63. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide includes SEQ ID NO:63. In some embodiments, the first chimeric polypeptide in the first multi-chain chimeric polypeptide includes SEQ ID NO:64. In some embodiments, the second chimeric polypeptide includes a sequence having at least 80% identity with SEQ ID NO:65. In some embodiments, the second chimeric polypeptide includes a sequence having at least 90% identity with SEQ ID NO:65. In some embodiments, the second chimeric polypeptide includes a sequence having at least 95% identity with SEQ ID NO:65. In some embodiments, the second chimeric polypeptide includes SEQ ID NO:65. In some embodiments, the second chimeric polypeptide includes SEQ ID NO:66. In some embodiments, the first target-binding domain and the linker domain of the first chimeric peptide in the second multi-chain chimeric peptide are directly adjacent to each other in the first chimeric peptide. In some embodiments, the first chimeric peptide in the second multi-chain chimeric peptide further includes a linker sequence between the first target-binding domain and the linker domain of the first chimeric peptide.In some embodiments, the linker domain and the first domain of the affinity domain pair are directly adjacent to each other in the first chimeric polypeptide of the second multi-chain chimeric polypeptide. In some embodiments, the first chimeric polypeptide of the second multi-chain chimeric polypeptide further includes a linker sequence between the linker domain and the first domain of the affinity domain pair in the first chimeric polypeptide. In some embodiments, the second domain of the affinity domain pair and the second target-binding domain of the second chimeric polypeptide of the second multi-chain chimeric polypeptide are directly adjacent to each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide of 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 in 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 having at least 80% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 90% identity with SEQ ID NO:5. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 95% identity with 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 having at least 80% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 90% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 95% identity with SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes SEQ ID NO:9. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 80% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 90% identity with SEQ ID NO:10. In some embodiments, the soluble human tissue factor domain includes a sequence having at least 95% identity with SEQ ID NO:3. In some embodiments, the soluble human tissue factor domain includes SEQ ID NO:10.In some embodiments, the soluble human tissue factor domain does not include one or more of the following: lysine at amino acid position 20 corresponding to amino acid position 22 corresponding to amino acid position 22 corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at amino acid position 45 corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at amino acid position 94 corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; arginine at amino acid position 135 corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the soluble human tissue factor domain does not include any of the following: lysine at amino acid position 20 corresponding to mature wild-type human tissue factor protein; isoleucine at amino acid position 22 corresponding to mature wild-type human tissue factor protein; tryptophan at amino acid position 45 corresponding to mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 corresponding to mature wild-type human tissue factor protein; tyrosine at amino acid position 94 corresponding to mature wild-type human tissue factor protein; arginine at amino acid position 135 corresponding to mature wild-type human tissue factor protein; and phenylalanine at amino acid position 140 corresponding to mature wild-type human tissue factor protein. In some embodiments, the soluble tissue factor domain cannot bind factor VIIa. In some embodiments, the soluble tissue factor domain does not convert inactive factor X into factor Xa. In some embodiments, the second multi-chain chimeric polypeptide does not stimulate coagulation in mammals. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide in the second multi-chain chimeric polypeptide further include 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 in the second multi-chain chimeric polypeptide is a sushi domain from the α chain (IL15Rα) of the human IL-15 receptor and soluble IL-15. In some embodiments, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, the soluble IL-15 includes a sequence having 80% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 includes a sequence having 90% identity with SEQ ID NO:26.In some embodiments, the soluble IL-15 comprises a sequence having 95% identity with SEQ ID NO:26. In some embodiments, the soluble IL-15 comprises SEQ ID NO:26. In some embodiments, the sushi domain of the IL15Rα comprises a sushi domain derived from human IL15Rα. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 80% identity with SEQ ID NO:24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 90% identity with SEQ ID NO:24. In some embodiments, the sushi domain derived from human IL15Rα comprises a sequence having 95% identity with 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 mature, full-length IL15Rα. In some embodiments, the affinity domain pairs in the second multi-chain chimeric polypeptide are selected from the group consisting of: barnase and barnstar, PKA and AKAP, an adaptor / docking tag module based on a mutant ribonuclease I fragment, and a SNARE module based on the interaction of protein synaptic fusion proteins, synapse-binding proteins, synaptic vesicle proteins, 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 having at least 80% identity with SEQ ID NO:22. In some embodiments, the soluble human IL-21 comprises a sequence having at least 90% identity with SEQ ID NO:22. In some embodiments, the soluble human IL-21 comprises a sequence having at least 95% identity with 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 includes soluble IL-7. 323. In some embodiments, the soluble IL-7 is soluble human IL-7. In some embodiments, the soluble human IL-7 includes a sequence having at least 80% identity with SEQ ID NO:23. In some embodiments, the soluble human IL-7 includes a sequence having at least 90% identity with SEQ ID NO:23. In some embodiments, the soluble human IL-7 includes a sequence having at least 95% identity with SEQ ID NO:23. In some embodiments, the soluble human IL-7 includes the sequence of SEQ ID NO:23. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide includes a sequence having at least 80% identity with SEQ ID NO:67. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide includes a sequence having at least 90% identity with SEQ ID NO:67. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide includes a sequence having at least 95% identity with 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 comprises SEQ ID NO:68. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 80% identity with SEQ ID NO:69. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO:69. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 95% identity with SEQ ID NO:69. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO:69. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO:70. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 80% identity with SEQ ID NO:71. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO:71. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 95% identity with SEQ ID NO:71. In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO:71.In some embodiments, the first chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO:72. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 80% identity with SEQ ID NO:73. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO:73. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises a sequence having at least 95% identity with SEQ ID NO:73. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO:73. In some embodiments, the second chimeric polypeptide in the second multi-chain chimeric polypeptide comprises SEQ ID NO:74. In some embodiments, the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the adapter domain. In some embodiments, the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the adapter domain.

[0027] As used herein, the term "chimera" refers to a polypeptide containing amino acid sequences (e.g., domains) originally derived from two different sources (e.g., two naturally occurring different proteins, such as those from the same or different species). For example, a chimeric polypeptide may contain domains derived from at least two naturally occurring different human proteins. In some instances, a chimeric polypeptide may contain a domain as a synthetic sequence (e.g., scFv) and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide may contain at least two different domains as synthetic sequences (e.g., two different scFvs).

[0028] "Multi-chain chimeric polypeptide" refers to a complex of two or more chimeric polypeptides.

[0029] An "antigen-binding domain" is one or more protein domains (e.g., formed from amino acids from a single polypeptide or from amino acids from two or more polypeptides (e.g., the same or different polypeptides)) capable of specifically binding to one or more different antigens. In some instances, an antigen-binding domain may bind to an antigen or epitope with similar specificity and affinity to naturally occurring antibodies. In some embodiments, the antigen-binding domain may be an antibody or a fragment thereof. In some embodiments, the antigen-binding domain may contain an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art.

[0030] "Soluble tissue factor domain" refers to a polypeptide that shares at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with a segment of a wild-type mammalian tissue factor protein (e.g., wild-type human tissue factor protein) lacking a transmembrane domain and an intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

[0031] The term "soluble interleukin protein" is used herein to refer to mature and secreted interleukin proteins or biologically active fragments thereof. In some instances, soluble interleukin proteins may contain sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with wild-type mature and secreted mammalian interleukin proteins (e.g., wild-type human interleukin protein) and retaining their biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

[0032] The term "soluble cytokine protein" is used herein to refer to mature and secreted cytokine proteins or biologically active fragments thereof. In some instances, soluble cytokine proteins may contain sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with wild-type mature and secreted mammalian interleukin proteins (e.g., wild-type human interleukin protein) and retaining their biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

[0033] The term "soluble interleukin receptor" is used herein in its broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) capable of binding one or more of its native ligands (e.g., under physiological conditions, such as at room temperature, in phosphate-buffered saline). For example, a soluble interleukin receptor may contain a sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with the extracellular domain of a wild-type interleukin receptor and retain its ability to specifically bind to one or more of its native ligands, but lacks its transmembrane domain (and optionally, also its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0034] The term "soluble cytokine receptor" is used herein in its broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) capable of binding one or more of its native ligands (e.g., under physiological conditions, such as at room temperature, in phosphate-buffered saline). For example, a soluble cytokine receptor may contain a sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with the extracellular domain of a wild-type cytokine receptor and retain its ability to specifically bind to one or more of its native receptors, but lacks its transmembrane domain (and optionally, also its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0035] The term "ligand of a costimulatory molecule" is used herein in its broadest sense to refer to a polypeptide capable of binding to and activating a costimulatory receptor molecule on an immune cell (e.g., under physiological conditions, such as at room temperature, in phosphate-buffered saline). For example, a ligand of a costimulatory molecule may comprise a sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with the ligand of the costimulatory molecule, which retains its ability to specifically bind to one or more of its native receptors. Non-limiting examples of ligands of costimulatory molecules are described herein.

[0036] The term "antibody" is used herein in the broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. Additional examples of antibodies are described herein. These additional examples of antibodies are known in the art.

[0037] “Affinity” refers to the strength of the sum of non-covalent interactions between an antigen-binding site and its binding partner (e.g., an antigen or epitope). Unless otherwise stated, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between an antigen-binding domain and a member of the antigen or epitope. The affinity of molecule X for its partner Y can be represented 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 methods known in the art, including those described herein. Affinity can be measured, for example, using surface plasmon resonance (SPR) techniques (e.g., ) or biolayer interferometry (e.g., This is determined by [method / method]. Additional methods for determining the affinity for antigen-binding domains and their corresponding antigens or epitopes are known in the art.

[0038] The term "a pair of affinity domains" refers to domains smaller than 1x10 -7 M (for example, less than 1x10) -8 M, less than 1x10 -9 M, less than 1x10 -10 M or less than 1x10 -11 M) of K D A pair of affinity domains are two distinct protein domains that bind specifically to each other. In some instances, an affinity domain pair may be a pair of naturally occurring proteins. In some embodiments, an affinity domain pair may be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs are described herein.

[0039] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen-binding domain. An epitope may consist, for example, of surface-accessible amino acid residues and / or sugar side chains, and may possess specific three-dimensional structural characteristics and specific charge properties. The difference between conformational epitopes and non-conformational epitopes is that binding to the former, but not to the latter, may be lost in the presence of denaturing solvents. Epitopes may include amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding. Methods for identifying epitopes that bind to antigen-binding domains are known in the art.

[0040] "Immune effector cells" are cells of the mammalian immune system that are capable of directly or indirectly recognizing pathogenic cells (e.g., cancer cells) in mammals and / or causing cell arrest or cell death in said cells. 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.

[0041] The term "treatment" refers to the improvement of at least one symptom of a disease. In some instances, the disease being treated is cancer, and improving at least one symptom of cancer includes reducing abnormal proliferation, gene expression, signal transduction, translation, and / or factor secretion. Generally, a treatment method involves administering a therapeutically effective amount of a composition to a subject who needs or has been identified as needing such treatment, said composition reducing at least one symptom of the disease.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, this specification (including definitions) shall prevail.

[0043] Other features and advantages of the invention will become apparent from the following detailed description and drawings, as well as from the claims. Attached Figure Description

[0044] Figure 1 A schematic diagram of an exemplary IL-12 / IL-15RαSu DNA construct is shown.

[0045] Figure 2 A schematic diagram of an exemplary IL-18 / TF / IL-15 DNA construct is shown.

[0046] Figure 3 A schematic diagram illustrating the interaction between exemplary IL-12 / IL-15RαSu and IL-18 / TF / IL-15 DNA constructs is shown.

[0047] Figure 4 A schematic diagram is shown of the interaction between an exemplary IL-12 / IL-15RαSu and IL-18 / TF / IL-15 fusion protein to generate an IL-18 / TF / IL-15:IL-12 / IL-15RαSu complex (18t15-12s).

[0048] Figure 5 A schematic diagram of an exemplary IL-7 / IL-15RαSu DNA construct is shown.

[0049] Figure 6 A schematic diagram of an exemplary IL-21 / TF / IL-15 DNA construct is shown.

[0050] Figure 7A schematic diagram illustrating the interaction between exemplary IL-7 / IL-15RαSu and IL-21 / TF / IL-15 DNA constructs is shown.

[0051] Figure 8 A schematic diagram is shown of the interaction between an exemplary IL-7 / IL-15RαSu and IL-21 / TF / IL-15 fusion protein to generate the IL-21 / TF / IL-15:IL-7 / IL-15RαSu complex (21t15-7s).

[0052] Figure 9 A schematic diagram of an exemplary IL-21 / IL-15RαSu DNA construct is shown.

[0053] Figure 10 A schematic diagram of an exemplary IL-7 / TF / IL-15 DNA construct is shown.

[0054] Figure 11 A schematic diagram illustrating the interaction between exemplary IL-21 / IL-15RαSu and IL-7 / TF / IL-15 DNA constructs is shown.

[0055] Figure 12 A schematic diagram is shown of the interaction between an exemplary IL-21 / IL-15RαSu and IL-7 / TF / IL-15 fusion protein to produce an IL-7 / TF / IL-15:IL-21 / IL-15RαSU complex (7t15-21s).

[0056] Figure 13 shows a graph illustrating the fold increase in STAT4 and STAT5 expression in unstimulated NK cells (US) or in NK cells exposed to an exemplary second multi-chain chimeric peptide and an exemplary IgG1 antibody construct (7t15-21s: anti-TF Ab), an exemplary first multi-chain chimeric peptide (18t15-12s), a combination of recombinant IL-12 and IL-18 (single cytokine), or recombinant IL-12 (IL-12) or recombinant IL-18 (IL-18) for 5 minutes, 15 minutes, 30 minutes, 60 minutes, or overnight.

[0057] Figure 14 This is a graph showing the extracellular acidification rate (ECAR) in unstimulated NK cells and in NK cells exposed to 10 nM or 100 nM of an exemplary first multi-chain chimeric polypeptide (18t15-12s) or in combination with recombinant cytokines (IL-12, IL-15, and IL-18) for 0 to 4 hours. The NK cells were obtained from three different donors.

[0058] Figure 15This is a graph showing the extracellular acidification rate (ECAR) in unstimulated NK cells and NK cells exposed to either an exemplary first multi-chain chimeric polypeptide (18t15-12s) or a combination of recombinant cytokines (IL-12, IL-15, and IL-18) for 0 to 3 hours. The NK cells were obtained from three different donors. The NK cells were further exposed to the indicated additions of glucose, oligomycin, and 2-deoxyglucose.

[0059] Figure 16 This is a fold change graph of gene expression of mTORC1, MYC, and SREBP in unstimulated NK cells or NK cells exposed to a combination of recombinant cytokines (IL-12, IL-15, and IL-18), an exemplary first multi-chain chimeric polypeptide (18t15-12s), or a control multi-chain chimeric polypeptide (7t15-21s) for 1 hour or 3 hours.

[0060] Figure 17 is a graph showing the expression of exemplary activation markers mTORC1, MYC, and SREBP in NK cells that were unstimulated or exposed to a combination of recombinant cytokines (IL-12, IL-15, and IL-18) or 100 nM of an exemplary first multi-chain chimeric polypeptide (18t15-12s) for 30 minutes to 3 hours.

[0061] Figure 18 The percentage of CD69-positive and interferon-γ-positive NK cells is shown after 3 hours or overnight stimulation of NK cells without stimulation or with a mixture of recombinant cytokines (IL-12, IL-15, and IL-18) or the exemplary first multi-chain chimeric polypeptide (18t15-12s).

[0062] Figure 19 shows the percentage of 41BB-positive and IL-21 receptor α-positive NK cells after 3 hours or overnight stimulation with no stimulation or with a mixture of recombinant cytokines (IL-12, IL-15, and IL-18) or the exemplary first multi-chain chimeric polypeptide (18t15-12s).

[0063] Figure 20The graph illustrates the fold expansion of NK cells over time after being treated with: (i) no initial stimulation followed by contact with an exemplary second multi-chain chimeric polypeptide (7t15-21s) and an exemplary IgG1 antibody construct (18t15-12s); (ii) initial stimulation with a combination of recombinant cytokines (IL-12, IL-15, and IL-18) followed by contact with a combination of an exemplary second multi-chain chimeric polypeptide (7t15-21s) and an exemplary IgG1 antibody construct (18t15-12s); or (iii) initial stimulation with an exemplary first multi-chain chimeric polypeptide followed by contact with a combination of an exemplary second multi-chain chimeric polypeptide (7t15-21s) and an exemplary IgG1 antibody construct (18t15-12s).

[0064] Figure 21 This is a graph showing the percentage of CD107a-positive cells treated with the following methods: (i) no initial stimulation followed by treatment with a combination of an exemplary second multi-chain chimeric peptide (7t15-21s) and an exemplary IgG1 antibody construct (αTF Ab); (ii) initial stimulation with a combination of cytokines (IL-12, IL-15, and IL-18), followed by treatment with a combination of an exemplary second multi-chain chimeric peptide (7t15-21s) and an exemplary IgG1 antibody construct (αTF Ab); and (iii) initial stimulation with an exemplary first multi-chain chimeric peptide (18t15-12s), followed by treatment with an exemplary second multi-chain chimeric peptide (7t15-21s) and an exemplary IgG1 antibody construct (αTF Ab). (iv) Treatment with a combination of (ab) cytokines (IL-12, IL-15 and IL-18) followed by treatment with recombinant IL-15; or (vi) initial stimulation with an exemplary first multi-chain chimeric polypeptide.

[0065] Figure 22 This is a graph illustrating the oxygen consumption rate (OCR) analysis of stimulated human NK cells, where the OCR analysis consists of four phases: basal respiration (no drug), ATP-related respiration / proton leakage (1.5 μM oligomycin), maximal respiration (2 μM FCCP), and reserve respiration (0.5 μM rotenone). The OCR graph is a combined representation of NK cells obtained from three donors. Stimulation of NK cells is shown and described in the corresponding examples.

[0066] Figure 23 This is a graph showing the levels of IFN-γ mRNA and NKG2D mRNA after stimulation with 18t15-12s or a combination of single cytokines, where cells are divided into short-term activated (180 minutes) or long-term activated (overnight stimulation 12-16 hours) groups, or unstimulated groups.

[0067] Figure 24 This demonstrates adoptive transfer of NK cells (CD56) expanded with 7t15-21s-αTF Ab in an NSG mouse model. + The persistence of cells was shown in the figure, where cells were not stimulated with culture medium as a control before expansion, or were stimulated with 100 nM 18t15-12s (kick) for 180 min or 12-16 h (overnight).

[0068] Figure 25 This diagram illustrates a "kick and expandation" epigenetic analysis of DNA methylation at CpG sites within the IFNγCNS-1 region. Cells were transferred to 24-well plates and stimulated with: no treatment, or with a mixture of single cytokines or a short exposure of 18t15-12s for 180 min or 12-16 h (overnight). NK cells were then washed and resuspended in complete RPMI 1640 medium supplemented with 7t15-21s plus anti-tissue factor antibody (αTF Ab) and incubated for 14 days (long-term). DNA isolated from the cells was then subjected to pyrosequencing analysis of DNA methylation at CpG sites within the IFNγCNS-1 region.

[0069] Figure 26 This diagram illustrates the epigenetic analysis of memory-like NK cells in vivo, where cells were stimulated for 12–16 hours with either a mixture of single cytokines or 18t15–12s. The NK cells were then washed and resuspended in complete RPMI 1640 medium supplemented with 7t15–21s + anti-tissue factor antibody (αTF Ab) and incubated for 14 days. After 14 days of expansion, the activated NK cells were processed for pyrosequencing analysis (pre-infusion samples) or injected into NSG mice (five groups, five mice per group). On day 9 post-infusion, spleens were collected from each mouse, and NK cells were purified and conjugated for each group (five groups, five mice per group). The samples (post-infusion) were then subjected to pyrosequencing analysis for DNA methylation at CpG sites located within the IFNγCNS-1 region.

[0070] Figure 27 This is a graph showing the fold increase of NK cells on day 5 of expansion, where the data shows that NK cells require IgG1 signaling for expansion.

[0071] Figure 28 This is an exemplary embodiment of the method currently claimed.

[0072] Figure 29 is a set of figures illustrating the expansion of primary NK cells using the methods described herein.

[0073] Figure 30 This is an exemplary illustration of a multi-chain chimeric polypeptide that can be used in the methods described herein.

[0074] Figure 31 This is a schematic diagram of an exemplary embodiment of the currently claimed method.

[0075] Figure 32 This is a schematic diagram of an exemplary embodiment of the currently claimed method.

[0076] Figure 33 It is a set of graphs showing the percentages of surface markers CD25 (top) and CD26 (bottom) after 10 days.

[0077] Figure 34 This is a set of graphs showing the percentages of surface markers NKp44 (top) and NKp30 (bottom) after 10 days.

[0078] Figure 35 This is a set of graphs showing the percentages of surface markers CD62L (top) and CD16 (bottom) after day 10. The data show that activation with 18t15-12s and amplification with 7t15-21s and anti-tissue factor IgG1 antibody lead to increased CD62L expression.

[0079] Figure 36 It shows the percentage of cytotoxicity in cells that are unstimulated or stimulated with a mixture of recombinant cytokines (IL-12, IL-15, and IL-18) or an exemplary first multi-chain chimeric polypeptide (18t15-12s).

[0080] Figure 37A This is a set of histograms showing the expansion of umbilical cord blood-derived NK cells, either untreated or stimulated with 18t15-12s (250 nM) for 180 min or 12-16 h followed by expansion with 7t15-21s (200 nM) and anti-tissue factor antibody (αTF Ab, 100 nM) for 7 days.

[0081] Figure 37B This is a graph showing the percentage of cell proliferation of umbilical cord blood-derived NK cells after untreated cells or after stimulation with 18t15-12s (250 nM) for 180 min or 12-16 h followed by expansion with 7t15-21s (200 nM) and anti-tissue factor antibody (αTF Ab, 100 nM) for 7 days. Detailed Implementation

[0082] This document provides specific combinations of two multi-chain chimeric peptides, the uses of said two multi-chain chimeric peptides, and kits comprising said two multi-chain chimeric peptides. The multi-chain chimeric peptides include a linker domain located 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 homologous IgG1 antibody (e.g., a monoclonal antibody). In some embodiments, the combination of the two multi-chain chimeric peptides described herein can be used to stimulate NK cells, induce or increase NK cell proliferation and / or induce NK cell differentiation, and said NK cells can subsequently be used to treat a subject.

[0083] This document provides a method for promoting the activation and proliferation of natural killer (NK) cells, the method comprising: (a) contacting the NK cells in a liquid culture medium (e.g., any liquid culture medium described herein) containing an effective amount of a first multi-chain chimeric polypeptide under conditions allowing NK cell differentiation, for a first time period; and (b) contacting the NK cells in a liquid culture medium containing an effective amount of (i) a second multi-chain chimeric polypeptide and (ii) an IgG1 antibody construct (e.g., any exemplary IgG1 antibody construct described herein) containing at least one antigen-binding domain specifically bound to the adapter domain, for a second time period, under conditions allowing the NK cells to activate and proliferate. NK cells generated by any of these methods are also provided, as well as methods for treating a subject with these NK cells. Compositions comprising any NK cells generated using these methods are also provided. A kit is also provided comprising: (a) a first multi-chain chimeric polypeptide comprising first and second chimeric polypeptides (e.g., any exemplary first multi-chain chimeric polypeptide described herein); (b) a second multi-chain chimeric polypeptide comprising the first and second chimeric polypeptides (e.g., any exemplary second multi-chain chimeric polypeptide described herein); and (c) an IgG1 antibody construct comprising at least one antigen-binding domain specifically bound to the adapter domain (e.g., any IgG1 antibody construct described herein). Figure 30-32 Exemplary embodiments of the methods provided herein are described in the document.

[0084] Current in vitro NK cell expansion methods may 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 for demonstrating effective irradiation of feeder cells; the need to deplete all feeder cell contaminants, including DNA, before the final product is administered to a patient; and the potentially tedious and expensive manufacturing process. Exemplary disadvantages of cytokine-based expansion methods include: poor purity of the final NK cell product, lower final NK cell counts compared to feeder cell expansion, the cost of recombinant cytokines, and the lack of commercially available GMP-grade cytokines (except IL-2). The methods described herein offer advantages such as the generation of highly activated NK cells from patients, large-scale expansion comparable to or similar to feeder cell methods, cost-effective expansion and activation with limited processing requirements, and the ability to inject cells into subjects, thereby allowing for in vivo cell expansion.

[0085] In some embodiments, the methods described herein can induce the generation and proliferation of NK cells that are highly activated and exhibit long-term persistence and high cytotoxicity to diseased cells in the recipient host (cells having any exemplary aspect of memory-like natural killer cells as described herein). In some embodiments of any of the methods described herein, the contact step of contacting NK cells with the 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).

[0086] In some embodiments, any of the methods described herein may result in an increase (e.g., before contacting NK cells with any of the exemplary first multi-chain chimeric polypeptides described herein) in one or more of the following rates compared to NK cells prior to implementing any of the methods described herein (e.g., before contacting NK cells with any of the exemplary first multi-chain chimeric polypeptides described herein): 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 5,000%). An increase of approximately 800%, an increase of approximately 1% to approximately 600%, an increase of approximately 1% to approximately 500%, an increase of approximately 1% to approximately 400%, an increase of approximately 1% to approximately 300%, an increase of approximately 1% to approximately 200%, an increase of approximately 1% to approximately 180%, an increase of approximately 1% to approximately 160%, an increase of approximately 1% to approximately 140%, an increase of approximately 1% to approximately 120%, an increase of approximately 1% to approximately 100%, an increase of approximately 1% to approximately 80%, an increase of approximately 1% to approximately 60%, an increase of approximately 1% to approximately 40%, an increase of approximately 1% to approximately 30%, an increase of approximately 1% to approximately 20%, and an increase of approximately 1% to... An increase of approximately 15%, an increase of approximately 1% to approximately 10%, an increase of approximately 1% to approximately 5%, an increase of approximately 5% to approximately 5,000%, an increase of approximately 5% to approximately 4,000%, an increase of approximately 5% to approximately 3,000%, an increase of approximately 5% to approximately 2,000%, an increase of approximately 5% to approximately 1,000%, an increase of approximately 5% to approximately 800%, an increase of approximately 5% to approximately 600%, an increase of approximately 5% to approximately 500%, an increase of approximately 5% to approximately 400%, an increase of approximately 5% to approximately 300%, an increase of approximately 5% to approximately 200%, an increase of approximately 5% to approximately 180%, and an increase of approximately 5% to approximately An increase of 160%, an increase of approximately 5% to approximately 140%, an increase of approximately 5% to approximately 120%, an increase of approximately 5% to approximately 100%, an increase of approximately 5% to approximately 80%, an increase of approximately 5% to approximately 60%, an increase of approximately 5% to approximately 40%, an increase of approximately 5% to approximately 30%, an increase of approximately 5% to approximately 20%, an increase of approximately 5% to approximately 15%, an increase of approximately 5% to approximately 10%, an increase of approximately 10% to approximately 5,000%, an increase of approximately 10% to approximately 4,000%, an increase of approximately 10% to approximately 3,000%, an increase of approximately 10% to approximately 2,000%, an increase of approximately 10% to approximately 1,An increase of 000%, an increase of approximately 10% to approximately 800%, an increase of approximately 10% to approximately 600%, an increase of approximately 10% to approximately 500%, an increase of approximately 10% to approximately 400%, an increase of approximately 10% to approximately 300%, an increase of approximately 10% to approximately 200%, an increase of approximately 10% to approximately 180%, an increase of approximately 10% to approximately 160%, an increase of approximately 10% to approximately 140%, an increase of approximately 10% to approximately 120%, an increase of approximately 10% to approximately 100%, an increase of approximately 10% to approximately 80%, an increase of approximately 10% to approximately 60%, an increase of approximately 10% to approximately 40%, an increase of approximately 10% to approximately 30%, and an increase of approximately 10% to approximately 20%. Increase, approximately 10% to approximately 15%, approximately 15% to approximately 5,000%, approximately 15% to approximately 4,000%, approximately 15% to approximately 3,000%, approximately 15% to approximately 2,000%, approximately 15% to approximately 1,000%, approximately 15% to approximately 800%, approximately 15% to approximately 600%, approximately 15% to approximately 500%, approximately 15% to approximately 400%, approximately 15% to approximately 300%, approximately 15% to approximately 200%, approximately 15% to approximately 180%, approximately 15% to approximately 160%, approximately 15% to approximately 140%, approximately 1 Increases of 5% to approximately 120%, 15% to approximately 100%, 15% to approximately 80%, 15% to approximately 60%, 15% to approximately 40%, 15% to approximately 30%, 15% to approximately 20%, 20% to approximately 5,000%, 20% to approximately 4,000%, 20% to approximately 3,000%, 20% to approximately 2,000%, 20% to approximately 1,000%, 20% to approximately 800%, 20% to approximately 600%, 20% to approximately 500%, and 20% to approximately 400%. Increase, approximately 20% to approximately 300% increase, approximately 20% to approximately 200% increase, approximately 20% to approximately 180% increase, approximately 20% to approximately 160% increase, approximately 20% to approximately 140% increase, approximately 20% to approximately 120% increase, approximately 20% to approximately 100% increase, approximately 20% to approximately 80% increase, approximately 20% to approximately 60% increase, approximately 20% to approximately 40% increase, approximately 20% to approximately 30% increase, approximately 30% to approximately 5,000% increase, approximately 30% to approximately 4,000% increase, approximately 30% to approximately 3,000% increase, approximately 30% to approximately 2,000% increase, approximately 30% to approximately 1,An increase of approximately 0.00%, an increase of approximately 30% to approximately 800%, an increase of approximately 30% to approximately 600%, an increase of approximately 30% to approximately 500%, an increase of approximately 30% to approximately 400%, an increase of approximately 30% to approximately 300%, an increase of approximately 30% to approximately 200%, an increase of approximately 30% to approximately 180%, an increase of approximately 30% to approximately 160%, an increase of approximately 30% to approximately 140%, an increase of approximately 30% to approximately 120%, an increase of approximately 30% to approximately 100%, an increase of approximately 30% to approximately 80%, an increase of approximately 30% to approximately 60%, an increase of approximately 30% to approximately 40%, an increase of approximately 40% to approximately 5,000%, an increase of approximately 40% to approximately 4,000%, and an increase of approximately 40% to approximately 3,000%. Increase, approximately 40% to approximately 2,000% increase, approximately 40% to approximately 1,000% increase, approximately 40% to approximately 800% increase, approximately 40% to approximately 600% increase, approximately 40% to approximately 500% increase, approximately 40% to approximately 400% increase, approximately 40% to approximately 300% increase, approximately 40% to approximately 200% increase, approximately 40% to approximately 180% increase, approximately 40% to approximately 160% increase, approximately 40% to approximately 140% increase, approximately 40% to approximately 120% increase, approximately 40% to approximately 100% increase, approximately 40% to approximately 80% increase, approximately 40% to approximately 60% increase, approximately 60% to approximately 5,000% increase, approximately 60% to approximately 4,000% increase An increase of approximately 60% to approximately 3,000%, an increase of approximately 60% to approximately 2,000%, an increase of approximately 60% to approximately 1,000%, an increase of approximately 60% to approximately 800%, an increase of approximately 60% to approximately 600%, an increase of approximately 60% to approximately 500%, an increase of approximately 60% to approximately 400%, an increase of approximately 60% to approximately 300%, an increase of approximately 60% to approximately 200%, an increase of approximately 60% to approximately 180%, an increase of approximately 60% to approximately 160%, an increase of approximately 60% to approximately 140%, an increase of approximately 60% to approximately 120%, an increase of approximately 60% to approximately 100%, an increase of approximately 60% to approximately 80%, an increase of approximately 80% to approximately 5,000%, and an increase of approximately 80% to approximately 4,000%. Increase, approximately 80% to approximately 3,000%, approximately 80% to approximately 2,000%, approximately 80% to approximately 1,000%, approximately 80% to approximately 800%, approximately 80% to approximately 600%, approximately 80% to approximately 500%, approximately 80% to approximately 400%, approximately 80% to approximately 300%, approximately 80% to approximately 200%, approximately 80% to approximately 180%, approximately 80% to approximately 160%, approximately 80% to approximately 140%, approximately 80% to approximately 120%, approximately 80% to approximately 100%, approximately 100% to approximately 5,000%, approximately 100% to approximately 4,000%, approximately 100% to approximately 3,An increase of approximately 100% to approximately 2,000%, an increase of approximately 100% to approximately 1,000%, an increase of approximately 100% to approximately 800%, an increase of approximately 100% to approximately 600%, an increase of approximately 100% to approximately 500%, an increase of approximately 100% to approximately 400%, an increase of approximately 100% to approximately 300%, an increase of approximately 100% to approximately 200%, an increase of approximately 100% to approximately 180%, an increase of approximately 100% to approximately 160%, an increase of approximately 100% to approximately 140%, an increase of approximately 100% to approximately 120%, an increase of approximately 120% to approximately 5,000%, an increase of approximately 120% to approximately 4,000%, an increase of approximately 120% to approximately 3,000%, and approximately 120%. Increases of approximately 120% to approximately 2,000%, approximately 120% to approximately 1,000%, approximately 120% to approximately 800%, approximately 120% to approximately 600%, approximately 120% to approximately 500%, approximately 120% to approximately 400%, approximately 120% to approximately 300%, approximately 120% to approximately 200%, approximately 120% to approximately 180%, approximately 120% to approximately 160%, approximately 120% to approximately 140%, approximately 140% to approximately 5,000%, approximately 140% to approximately 4,000%, approximately 140% to approximately 3,000%, approximately 140% to approximately 2,000%, and approximately 140% to approximately 1,000%. Increase, approximately 140% to approximately 800%, approximately 140% to approximately 600%, approximately 140% to approximately 500%, approximately 140% to approximately 400%, approximately 140% to approximately 300%, approximately 140% to approximately 200%, approximately 140% to approximately 180%, approximately 140% to approximately 160%, approximately 160% to approximately 5,000%, approximately 160% to approximately 4,000%, approximately 160% to approximately 3,000%, approximately 160% to approximately 2,000%, approximately 160% to approximately 1,000%, approximately 160% to approximately 800%, approximately 160% to approximately 600%, approximately 160% to approximately 500%. The increase, approximately 160% to approximately 400%, approximately 160% to approximately 300%, approximately 160% to approximately 200%, approximately 160% to approximately 180%, approximately 180% to approximately 5,000%, approximately 180% to approximately 4,000%, approximately 180% to approximately 3,000%, approximately 180% to approximately 2,000%, approximately 180% to approximately 1,000%, approximately 180% to approximately 800%, approximately 180% to approximately 600%, approximately 180% to approximately 500%, approximately 180% to approximately 400%, approximately 180% to approximately 300%, approximately 180% to approximately 200%, approximately 200% to approximately 5,An increase of approximately 0.00%, an increase of approximately 200% to approximately 4,000%, an increase of approximately 200% to approximately 3,000%, an increase of approximately 200% to approximately 2,000%, an increase of approximately 200% to approximately 1,000%, an increase of approximately 200% to approximately 800%, an increase of approximately 200% to approximately 600%, an increase of approximately 200% to approximately 500%, an increase of approximately 200% to approximately 400%, an increase of approximately 200% to approximately 300%, an increase of approximately 300% to approximately 5,000%, an increase of approximately 300% to approximately 4,000%, an increase of approximately 300% to approximately 3,000%, an increase of approximately 300% to approximately 2,000%, an increase of approximately 300% to approximately 1,000%. An increase of approximately 0.00%, an increase of approximately 300% to approximately 800%, an increase of approximately 300% to approximately 600%, an increase of approximately 300% to approximately 500%, an increase of approximately 300% to approximately 400%, an increase of approximately 400% to approximately 500%, an increase of approximately 400% to approximately 500%, an increase of approximately 400% to approximately 4000%, an increase of approximately 400% to approximately 3000%, an increase of approximately 400% to approximately 2000%, an increase of approximately 400% to approximately 1000%, an increase of approximately 400% to approximately 800%, an increase of approximately 400% to approximately 600%, an increase of approximately 400% to approximately 500%, an increase of approximately 500% to approximately 5000%, and an increase of approximately 500% to approximately 4000%. Increase of approximately 500% to approximately 3,000%, approximately 500% to approximately 2,000%, approximately 500% to approximately 1,000%, approximately 500% to approximately 800%, approximately 500% to approximately 600%, approximately 600% to approximately 5,000%, approximately 600% to approximately 4,000%, approximately 600% to approximately 3,000%, approximately 600% to approximately 2,000%, approximately 600% to approximately 1,000%, approximately 600% to approximately 800%, approximately 800% to approximately 5,000%, approximately 800% to approximately 4,000%, approximately 800% to approximately 3,000%. An increase of approximately 800% to approximately 2,000%, an increase of approximately 800% to approximately 1,000%, an increase of approximately 1,000% to approximately 5,000%, an increase of approximately 1,000% to approximately 4,000%, an increase of approximately 1,000% to approximately 3,000%, an increase of approximately 1,000% to approximately 2,000%, an increase of approximately 2,000% to approximately 5,000%, an increase of approximately 2,000% to approximately 4,000%, an increase of approximately 2,000% to approximately 3,000%, an increase of approximately 3,000% to approximately 5,000%, an increase of approximately 3,000% to approximately 4,000%, or an increase of approximately 4,000% to approximately 5,000%.

[0087] In some embodiments of any of the methods described herein, the method results in an increase in the number of live NK cells, for example, about 5 to about 5,000 times, about 5 to about 4,500 times, about 5 to about 4,000 times, about 5 to about 3,500 times, about 5 to about 3,000 times, about 5 to about 2,500 times, about 5 to about 2,000 times, about 5 to about 1,500 times, about 5 to about 1,000 times, about 5 to about 800 times, about 5 to about 600 times, about 5 to about 500 times, about 5 to about 450 times, about 5 to about 400 times, about 5 to about 350 times, about 5 times, and about 5 times, compared to the number of NK cells prior to the step of contacting NK cells with any of the exemplary first multi-chain chimeric polypeptides described herein. From approximately 300 times, approximately 5 times to approximately 250 times, approximately 5 times to approximately 200 times, approximately 5 times to approximately 150 times, approximately 5 times to approximately 100 times, approximately 5 times to approximately 80 times, approximately 5 times to approximately 60 times, approximately 5 times to approximately 40 times, approximately 5 times to approximately 20 times, approximately 5 times to approximately 10 times, approximately 10 times to approximately 5,000 times, approximately 10 times to approximately 4,500 times, approximately 10 times to approximately 4,000 times, approximately 10 times to approximately 3,500 times, approximately 10 times to approximately 3,000 times, approximately 10 times to approximately 2,500 times, approximately 10 times to approximately 2,000 times, approximately 10 times to approximately 1,500 times, approximately 10 times to approximately 1,000 times, approximately 10 times to approximately 800 times, approximately 10 times to approximately 600 times, approximately 10 times to approximately 500 times, approximately 10 times to approximately 4 50 times, approximately 10 times to approximately 400 times, approximately 10 times to approximately 350 times, approximately 10 times to approximately 300 times, approximately 10 times to approximately 250 times, approximately 10 times to approximately 200 times, approximately 10 times to approximately 150 times, approximately 10 times to approximately 100 times, approximately 10 times to approximately 80 times, approximately 10 times to approximately 60 times, approximately 10 times to approximately 40 times, approximately 10 times to approximately 20 times, approximately 20 times To approximately 5,000 times, approximately 20 times to approximately 4,500 times, approximately 20 times to approximately 4,000 times, approximately 20 times to approximately 3,500 times, approximately 20 times to approximately 3,000 times, approximately 20 times to approximately 2,500 times, approximately 20 times to approximately 2,000 times, approximately 20 times to approximately 1,500 times, approximately 20 times to approximately 1,000 times, approximately 20 times to approximately 800 times, approximately 20 times to approximately 600 times, approximately 20 times to approximately 500 times, approximately 20 times to approximately 450 times, approximately 20 times to approximately 400 times, approximately 20 times to approximately 350 times, approximately 20 times to approximately 300 times, approximately 20 times to approximately 250 times, approximately 20 times to approximately 200 times, approximately 20 times to approximately 150 times, approximately 20 times to approximately 100 times, approximately 20 times to approximately 80 times, approximately 20 times to approximately 60 times, approximately 20 times to approximately 40 times, approximately 40 times to approximately 5,000 times, approximately 40 times to approximately 4,500 times, approximately 40 times to approximately 4,000 times, approximately 40 times to approximately 3,500 times, approximately 40 times to approximately 3,000 times, approximately 40 times to approximately 2,500 times, approximately 40 times to approximately 2,000 times, approximately 40 times to approximately 1,500 times, approximately 40 times to approximately 1,000 times, approximately 40 times to approximately 800 times, approximately 40 times to approximately 600 times, approximately 40 times to approximately 500 times, approximately 40 times to approximately 450 times, approximately 40 times to approximately 400 times, approximately 40 times to approximately 350 times, approximately 40 times to approximately 300 times, approximately 40 times to approximately 250 times, approximately 40 times to approximately 200 times, approximately 40 times to approximately 150 times, approximately 40 times to approximately 100 times Approximately 40 to 80 times, approximately 40 to 60 times, approximately 60 to 5,000 times, approximately 60 to 4,500 times, approximately 60 to 4,000 times, approximately 60 to 3,500 times, approximately 60 to 3,000 times, approximately 60 to 2,500 times, approximately 60 to 2,000 times, approximately 60 to 1,500 times. Approximately 60 to approximately 1,000 times, approximately 60 to approximately 800 times, approximately 60 to approximately 600 times, approximately 60 to approximately 500 times, approximately 60 to approximately 450 times, approximately 60 to approximately 400 times, approximately 60 to approximately 350 times, approximately 60 to approximately 300 times, approximately 60 to approximately 250 times, approximately 60 to approximately 200 times, approximately 60 to approximately 150 times, approximately 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 approximately 1,000 times, approximately 80 times to approximately 800 times, approximately 80 times to approximately 600 times, approximately 80 times to approximately 500 times, approximately 80 times to approximately 450 times, approximately 80 times to approximately 400 times, approximately 80 times to approximately 350 times, approximately 80 times to approximately 300 times, approximately 80 times to approximately 250 times, approximately 80 times to approximately 200 times, approximately 80 times to approximately 150 times, approximately 80 From approximately 100 times to approximately 5,000 times, from approximately 100 times to approximately 4,500 times, from approximately 100 times to approximately 4,000 times, from approximately 100 times to approximately 3,500 times, from approximately 100 times to approximately 3,000 times, from approximately 100 times to approximately 2,500 times, from approximately 100 times to approximately 2,000 times, from approximately 100 times to approximately 1,500 times, from approximately 100 times... To approximately 1,000 times, approximately 100 times to approximately 800 times, approximately 100 times to approximately 600 times, approximately 100 times to approximately 500 times, approximately 100 times to approximately 450 times, approximately 100 times to approximately 400 times, approximately 100 times to approximately 350 times, approximately 100 times to approximately 300 times, approximately 100 times to approximately 250 times, approximately 100 times to approximately 200 times, approximately 100 times to approximately 1 50 times, approximately 150 times to approximately 5,000 times, approximately 150 times to approximately 4,500 times, approximately 150 times to approximately 4,000 times, approximately 150 times to approximately 3,500 times, approximately 150 times to approximately 3,000 times, approximately 150 times to approximately 2,500 times, approximately 150 times to approximately 2,000 times, approximately 150 times to approximately 1,500 times, approximately 150 times to approximately 1,000 times, approximately 150 times to approximately 800 times, approximately 150 times to approximately 600 times, approximately 150 times to approximately 500 times, approximately 150 times to approximately 450 times, approximately 150 times to approximately 400 times, approximately 150 times to approximately 350 times, approximately 150 times to approximately 300 times, approximately 150 times to approximately 250 times, approximately 150 times to approximately 200 times, approximately 200 times to approximately 5,000 times. 0x, approximately 200x to approximately 4,500x, approximately 200x to approximately 4,000x, approximately 200x to approximately 3,500x, approximately 200x to approximately 3,000x, approximately 200x to approximately 2,500x, approximately 200x to approximately 2,000x, approximately 200x to approximately 1,500x, approximately 200x to approximately 1,000x, approximately 200x to approximately 80x 0x, approximately 200x to approximately 600x, approximately 200x to approximately 500x, approximately 200x to approximately 450x, approximately 200x to approximately 400x, approximately 200x to approximately 350x, approximately 200x to approximately 300x, approximately 200x to approximately 250x, approximately 250x to approximately 5,000x, approximately 250x to approximately 4,500x, approximately 250x to approximately 4, 000 times, approximately 250 times to approximately 3,500 times, approximately 250 times to approximately 3,000 times, approximately 250 times to approximately 2,500 times, approximately 250 times to approximately 2,000 times, approximately 250 times to approximately 1,500 times, approximately 250 times to approximately 1,000 times, approximately 250 times to approximately 800 times, approximately 250 times to approximately 600 times, approximately 250 times to approximately 500 times. Approximately 250 to 450 times, approximately 250 to 400 times, approximately 250 to 350 times, approximately 250 to 300 times, approximately 300 to 5,000 times, approximately 300 to 4,500 times, approximately 300 to 4,000 times, approximately 300 to 3,500 times, approximately 300 to 3,000 times, approximately 300 to Approximately 2,500 times, approximately 300 times to approximately 2,000 times, approximately 300 times to approximately 1,500 times, approximately 300 times to approximately 1,000 times, approximately 300 times to approximately 800 times, approximately 300 times to approximately 600 times, approximately 300 times to approximately 500 times, approximately 300 times to approximately 450 times, approximately 300 times to approximately 400 times, approximately 300 times to approximately 350 times, approximately 3 50 times to approximately 5,000 times, approximately 350 times to approximately 4,500 times, approximately 350 times to approximately 4,000 times, approximately 350 times to approximately 3,500 times, approximately 350 times to approximately 3,000 times, approximately 350 times to approximately 2,500 times, approximately 350 times to approximately 2,000 times, approximately 350 times to approximately 1,500 times, approximately 350 times to approximately 1,000 times. Approximately 350 to approximately 800 times, approximately 350 to approximately 600 times, approximately 350 to approximately 500 times, approximately 350 to approximately 450 times, approximately 350 to approximately 400 times, approximately 400 to approximately 5,000 times, approximately 400 to approximately 4,500 times, approximately 400 to approximately 4,000 times, approximately 400 to approximately 3,500 times, approximately 400 to approximately 3,000 times, approximately 400 times to approximately 2,500 times, approximately 400 times to approximately 2,000 times, approximately 400 times to approximately 1,500 times, approximately 400 times to approximately 1,000 times, approximately 400 times to approximately 800 times, approximately 400 times to approximately 600 times, approximately 400 times to approximately 500 times, approximately 400 times to approximately 450 times, approximately 450 times to approximately 5,000 times, approximately 4 50 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 45 0x to approximately 600x, approximately 450x to approximately 500x, approximately 500x to approximately 5,000x, approximately 500x to approximately 4,500x, approximately 500x to approximately 4,000x, approximately 500x to approximately 3,500x, approximately 500x to approximately 3,000x, approximately 500x to approximately 2,500x, approximately 500x to approximately 2,000x, approximately 500x to... Approximately 1,500 times, approximately 500 to approximately 1,000 times, approximately 500 to approximately 800 times, approximately 500 to approximately 600 times, approximately 600 to approximately 5,000 times, approximately 600 to approximately 4,500 times, approximately 600 to approximately 4,000 times, approximately 600 to approximately 3,500 times, approximately 600 to approximately 3,000 times, approximately 600 to approximately 2, 500 times, approximately 600 times to approximately 2,000 times, approximately 600 times to approximately 1,500 times, approximately 600 times to approximately 1,000 times, approximately 600 times to approximately 800 times, approximately 800 times to approximately 5,000 times, approximately 800 times to approximately 4,500 times, approximately 800 times to approximately 4,000 times, approximately 800 times to approximately 3,500 times, approximately 800 times to approximately 3,000 times. 00 times, approximately 800 times to approximately 2,500 times, approximately 800 times to approximately 2,000 times, approximately 800 times to approximately 1,500 times, approximately 800 times to approximately 1,000 times, approximately 1,000 times to approximately 5,000 times, approximately 1,000 times to approximately 4,500 times, approximately 1,000 times to approximately 4,000 times, approximately 1,000 times to approximately 3,500 times, approximately 1,000 to approximately 3,000 times, approximately 1,000 to approximately 2,500 times, approximately 1,000 to approximately 2,000 times, approximately 1,000 to approximately 1,500 times, approximately 1,500 to approximately 5,000 times, approximately 1,500 to approximately 4,500 times, approximately 1,500 to approximately 4,000 times, approximately 1,500 to approximately 3,500 times. Approximately 1,500 to 3,000 times, approximately 1,500 to 2,500 times, approximately 1,500 to 2,000 times, approximately 2,000 to 5,000 times, approximately 2,000 to 4,500 times, approximately 2,000 to 4,000 times, approximately 2,000 to 3,500 times, approximately 2,000 to 3,000 times.000 times, approximately 2,000 to approximately 2,500 times, approximately 2,500 to approximately 5,000 times, approximately 2,500 to approximately 4,500 times, approximately 2,500 to approximately 4,000 times, approximately 2,500 to approximately 3,500 times, approximately 2,500 to approximately 3,000 times, approximately 3,000 to approximately 5,000 times, approximately 3,000 to approximately 4,500 times. 0x, approximately 3,000x to approximately 4,000x, approximately 3,000x to approximately 3,500x, approximately 3,500x to approximately 5,000x, approximately 3,500x to approximately 4,500x, approximately 3,500x to approximately 4,000x, approximately 4,000x to approximately 5,000x, approximately 4,000x to approximately 4,500x, or approximately 4,500x to approximately 5,000x.

[0088] In some embodiments, the method provides NK cells with improved persistence and cytotoxicity against diseased cells in a subject compared to untreated control NK cells.

[0089] Multi-chain chimeric peptide

[0090] The methods described in this article involve the use of two different multi-chain chimeric peptides.

[0091] The first multi-chain chimeric polypeptide provided herein comprises: (a) a first chimeric polypeptide comprising: a first target-binding domain; a soluble tissue factor domain; and a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising: a second domain of a pair of affinity domains; and a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first and second domains of the affinity domain pair, and the first target-binding domain and the second target-binding domain each independently and specifically bind to an IL-18 receptor or an IL-12 receptor.

[0092] The second multi-chain chimeric polypeptide provided herein comprises: (a) a first chimeric polypeptide comprising: a first target-binding domain, a linker domain, and a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising a second domain of a pair of affinity domains and a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first and second domains of the affinity domain pair, and the first target-binding domain and the second target-binding domain each independently and specifically bind to either the IL-7 receptor or the IL-21 receptor.

[0093] In some examples of any first and second multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide may each independently be from about 50 amino acids to about 3000 amino acids, from about 50 amino acids to about 2500 amino acids, from about 50 amino acids to about 2000 amino acids, from about 50 amino acids to about 1500 amino acids, from about 50 amino acids to about 1000 amino acids, from about 50 amino acids to about 950 amino acids, from about 50 amino acids to about 900 amino acids, from about 50 amino acids to about 850 amino acids, from about 50 amino acids to about 800 amino acids, from about 50 amino acids to about 750 amino acids, from about 50 amino acids to about 700 amino acids, and so on. From 50 amino acids to approximately 650 amino acids, from approximately 50 amino acids to approximately 600 amino acids, from approximately 50 amino acids to approximately 550 amino acids, from approximately 50 amino acids to approximately 500 amino acids, from approximately 50 amino acids to approximately 480 amino acids, from approximately 50 amino acids to approximately 460 amino acids, from approximately 50 amino acids to approximately 440 amino acids, from approximately 50 amino acids to approximately 420 amino acids, from approximately 50 amino acids to approximately 400 amino acids, from approximately 50 amino acids to approximately 380 amino acids, from approximately 50 amino acids to approximately 360 amino acids, from approximately 50 amino acids to approximately 340 amino acids, from approximately 50 amino acids to approximately 320 amino acids, from approximately 50 amino acids to approximately 300 amino acids, from approximately 50 amino acids to approximately... 280 amino acids, approximately 50 amino acids to approximately 260 amino acids, approximately 50 amino acids to approximately 240 amino acids, approximately 50 amino acids to approximately 220 amino acids, approximately 50 amino acids to approximately 200 amino acids, approximately 50 amino acids to approximately 150 amino acids, approximately 50 amino acids to approximately 100 amino acids, approximately 100 amino acids to approximately 3000 amino acids, approximately 100 amino acids to approximately 2500 amino acids, approximately 100 amino acids to approximately 2000 amino acids, approximately 100 amino acids to approximately 1500 amino acids, approximately 100 amino acids to approximately 1000 amino acids, approximately 100 amino acids to approximately 950 amino acids, approximately 100 amino acids to approximately 900 amino acids, approximately 100 amino acids... From approximately 850 amino acids, from approximately 100 amino acids to approximately 800 amino acids, from approximately 100 amino acids to approximately 750 amino acids, from approximately 100 amino acids to approximately 700 amino acids, from approximately 100 amino acids to approximately 650 amino acids, from approximately 100 amino acids to approximately 600 amino acids, from approximately 100 amino acids to approximately 550 amino acids, from approximately 100 amino acids to approximately 500 amino acids, from approximately 100 amino acids to approximately 480 amino acids, from approximately 100 amino acids to approximately 460 amino acids, from approximately 100 amino acids to approximately 440 amino acids, from approximately 100 amino acids to approximately 420 amino acids, from approximately 100 amino acids to approximately 400 amino acids, from approximately 100 amino acids to approximately 380 amino acids.Approximately 100 amino acids to approximately 360 amino acids, approximately 100 amino acids to approximately 340 amino acids, approximately 100 amino acids to approximately 320 amino acids, approximately 100 amino acids to approximately 300 amino acids, approximately 100 amino acids to approximately 280 amino acids, approximately 100 amino acids to approximately 260 amino acids, approximately 100 amino acids to approximately 240 amino acids, approximately 100 amino acids to approximately 220 amino acids, approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 150 amino acids, approximately 150 amino acids to approximately 3000 amino acids, approximately 150 amino acids to approximately 2500 amino acids, approximately 150 amino acids to approximately 2000 amino acids, approximately 150 amino acids to approximately 150 0 amino acids, approximately 150 amino acids to approximately 1000 amino acids, approximately 150 amino acids to approximately 950 amino acids, approximately 150 amino acids to approximately 900 amino acids, approximately 150 amino acids to approximately 850 amino acids, approximately 150 amino acids to approximately 800 amino acids, approximately 150 amino acids to approximately 750 amino acids, approximately 150 amino acids to approximately 700 amino acids, approximately 150 amino acids to approximately 650 amino acids, approximately 150 amino acids to approximately 600 amino acids, approximately 150 amino acids to approximately 550 amino acids, approximately 150 amino acids to approximately 500 amino acids, approximately 150 amino acids to approximately 480 amino acids, approximately 150 amino acids to approximately 460 amino acids, approximately 150 amino acids to Approximately 440 amino acids, approximately 150 to approximately 420 amino acids, approximately 150 to approximately 400 amino acids, approximately 150 to approximately 380 amino acids, approximately 150 to approximately 360 amino acids, approximately 150 to approximately 340 amino acids, approximately 150 to approximately 320 amino acids, approximately 150 to approximately 300 amino acids, approximately 150 to approximately 280 amino acids, approximately 150 to approximately 260 amino acids, approximately 150 to approximately 240 amino acids, approximately 150 to approximately 220 amino acids, approximately 150 to approximately 200 amino acids, approximately 200 to approximately 3000 amino acids, approximately 200 amino acids. From approximately 2500 amino acids, from approximately 200 amino acids to approximately 2000 amino acids, from approximately 200 amino acids to approximately 1500 amino acids, from approximately 200 amino acids to approximately 1000 amino acids, from approximately 200 amino acids to approximately 950 amino acids, from approximately 200 amino acids to approximately 900 amino acids, from approximately 200 amino acids to approximately 850 amino acids, from approximately 200 amino acids to approximately 800 amino acids, from approximately 200 amino acids to approximately 750 amino acids, from approximately 200 amino acids to approximately 700 amino acids, from approximately 200 amino acids to approximately 650 amino acids, from approximately 200 amino acids to approximately 600 amino acids, from approximately 200 amino acids to approximately 550 amino acids, from approximately 200 amino acids to approximately 500 amino acids.Approximately 200 to approximately 480 amino acids, approximately 200 to approximately 460 amino acids, approximately 200 to approximately 440 amino acids, approximately 200 to approximately 420 amino acids, approximately 200 to approximately 400 amino acids, approximately 200 to approximately 380 amino acids, approximately 200 to approximately 360 amino acids, approximately 200 to approximately 340 amino acids, approximately 200 to approximately 320 amino acids, approximately 200 to approximately 300 amino acids, approximately 200 to approximately 280 amino acids, approximately 200 to approximately 260 amino acids, approximately 200 to approximately 240 amino acids, approximately 200 to approximately 220 amino acids. Acids, approximately 220 amino acids to approximately 3000 amino acids, approximately 220 amino acids to approximately 2500 amino acids, approximately 220 amino acids to approximately 2000 amino acids, approximately 220 amino acids to approximately 1500 amino acids, approximately 220 amino acids to approximately 1000 amino acids, approximately 220 amino acids to approximately 950 amino acids, approximately 220 amino acids to approximately 900 amino acids, approximately 220 amino acids to approximately 850 amino acids, approximately 220 amino acids to approximately 800 amino acids, approximately 220 amino acids to approximately 750 amino acids, approximately 220 amino acids to approximately 700 amino acids, approximately 220 amino acids to approximately 650 amino acids, approximately 220 amino acids to approximately 600 amino acids, approximately 220 amino acids to Approximately 550 amino acids, approximately 220 amino acids to approximately 500 amino acids, approximately 220 amino acids to approximately 480 amino acids, approximately 220 amino acids to approximately 460 amino acids, approximately 220 amino acids to approximately 440 amino acids, approximately 220 amino acids to approximately 420 amino acids, approximately 220 amino acids to approximately 400 amino acids, approximately 220 amino acids to approximately 380 amino acids, approximately 220 amino acids to approximately 360 amino acids, approximately 220 amino acids to approximately 340 amino acids, approximately 220 amino acids to approximately 320 amino acids, approximately 220 amino acids to approximately 300 amino acids, approximately 220 amino acids to approximately 280 amino acids, approximately 220 amino acids to approximately 260 amino acids, approximately 220 amino acids... From approximately 240 amino acids, from approximately 240 amino acids to approximately 3000 amino acids, from approximately 240 amino acids to approximately 2500 amino acids, from approximately 240 amino acids to approximately 2000 amino acids, from approximately 240 amino acids to approximately 1500 amino acids, from approximately 240 amino acids to approximately 1000 amino acids, from approximately 240 amino acids to approximately 950 amino acids, from approximately 240 amino acids to approximately 900 amino acids, from approximately 240 amino acids to approximately 850 amino acids, from approximately 240 amino acids to approximately 800 amino acids, from approximately 240 amino acids to approximately 750 amino acids, from approximately 240 amino acids to approximately 700 amino acids, from approximately 240 amino acids to approximately 650 amino acids, from approximately 240 amino acids to approximately 600 amino acids.Approximately 240 amino acids to approximately 550 amino acids, approximately 240 amino acids to approximately 500 amino acids, approximately 240 amino acids to approximately 480 amino acids, approximately 240 amino acids to approximately 460 amino acids, approximately 240 amino acids to approximately 440 amino acids, approximately 240 amino acids to approximately 420 amino acids, approximately 240 amino acids to approximately 400 amino acids, approximately 240 amino acids to approximately 380 amino acids, approximately 240 amino acids to approximately 360 amino acids, approximately 240 amino acids to approximately 340 amino acids, approximately 240 amino acids to approximately 320 amino acids, approximately 240 amino acids to approximately 300 amino acids, approximately 240 amino acids to approximately 280 amino acids, approximately 240 amino acids to approximately 260 amino acids. Acids, approximately 260 amino acids to approximately 3000 amino acids, approximately 260 amino acids to approximately 2500 amino acids, approximately 260 amino acids to approximately 2000 amino acids, approximately 260 amino acids to approximately 1500 amino acids, approximately 260 amino acids to approximately 1000 amino acids, approximately 260 amino acids to approximately 950 amino acids, approximately 260 amino acids to approximately 900 amino acids, approximately 260 amino acids to approximately 850 amino acids, approximately 260 amino acids to approximately 800 amino acids, approximately 260 amino acids to approximately 750 amino acids, approximately 260 amino acids to approximately 700 amino acids, approximately 260 amino acids to approximately 650 amino acids, approximately 260 amino acids to approximately 600 amino acids, approximately 260 amino acids to Approximately 550 amino acids, approximately 260 amino acids to approximately 500 amino acids, approximately 260 amino acids to approximately 480 amino acids, approximately 260 amino acids to approximately 460 amino acids, approximately 260 amino acids to approximately 440 amino acids, approximately 260 amino acids to approximately 420 amino acids, approximately 260 amino acids to approximately 400 amino acids, approximately 260 amino acids to approximately 380 amino acids, approximately 260 amino acids to approximately 360 amino acids, approximately 260 amino acids to approximately 340 amino acids, approximately 260 amino acids to approximately 320 amino acids, approximately 260 amino acids to approximately 300 amino acids, approximately 260 amino acids to approximately 280 amino acids, approximately 280 amino acids to approximately 3000 amino acids, approximately 280 amino acids From approximately 2500 amino acids, from approximately 280 amino acids to approximately 2000 amino acids, from approximately 280 amino acids to approximately 1500 amino acids, from approximately 280 amino acids to approximately 1000 amino acids, from approximately 280 amino acids to approximately 950 amino acids, from approximately 280 amino acids to approximately 900 amino acids, from approximately 280 amino acids to approximately 850 amino acids, from approximately 280 amino acids to approximately 800 amino acids, from approximately 280 amino acids to approximately 750 amino acids, from approximately 280 amino acids to approximately 700 amino acids, from approximately 280 amino acids to approximately 650 amino acids, from approximately 280 amino acids to approximately 600 amino acids, from approximately 280 amino acids to approximately 550 amino acids, from approximately 280 amino acids to approximately 500 amino acids.From approximately 280 amino acids to approximately 480 amino acids, from approximately 280 amino acids to approximately 460 amino acids, from approximately 280 amino acids to approximately 440 amino acids, from approximately 280 amino acids to approximately 420 amino acids, from approximately 280 amino acids to approximately 400 amino acids, from approximately 280 amino acids to approximately 380 amino acids, from approximately 280 amino acids to approximately 360 amino acids, from approximately 280 amino acids to approximately 340 amino acids, from approximately 280 amino acids to approximately 320 amino acids, from approximately 280 amino acids to approximately 300 amino acids, from approximately 300 amino acids to approximately 3000 amino acids, from approximately 300 amino acids to approximately 2500 amino acids, from approximately 300 amino acids to approximately 2000 amino acids, from approximately 300 amino acids to approximately 150 amino acids. 0 amino acids, approximately 300 amino acids to approximately 1000 amino acids, approximately 300 amino acids to approximately 950 amino acids, approximately 300 amino acids to approximately 900 amino acids, approximately 300 amino acids to approximately 850 amino acids, approximately 300 amino acids to approximately 800 amino acids, approximately 300 amino acids to approximately 750 amino acids, approximately 300 amino acids to approximately 700 amino acids, approximately 300 amino acids to approximately 650 amino acids, approximately 300 amino acids to approximately 600 amino acids, approximately 300 amino acids to approximately 550 amino acids, approximately 300 amino acids to approximately 500 amino acids, approximately 300 amino acids to approximately 480 amino acids, approximately 300 amino acids to approximately 460 amino acids, approximately 300 amino acids to Approximately 440 amino acids, approximately 300 to approximately 420 amino acids, approximately 300 to approximately 400 amino acids, approximately 300 to approximately 380 amino acids, approximately 300 to approximately 360 amino acids, approximately 300 to approximately 340 amino acids, approximately 300 to approximately 320 amino acids, approximately 320 to approximately 3000 amino acids, approximately 320 to approximately 2500 amino acids, approximately 320 to approximately 2000 amino acids, approximately 320 to approximately 1500 amino acids, approximately 320 to approximately 1000 amino acids, approximately 320 to approximately 950 amino acids, approximately 320 to approximately 900 amino acids, approximately 3 From 20 amino acids to approximately 850 amino acids, from approximately 320 amino acids to approximately 800 amino acids, from approximately 320 amino acids to approximately 750 amino acids, from approximately 320 amino acids to approximately 700 amino acids, from approximately 320 amino acids to approximately 650 amino acids, from approximately 320 amino acids to approximately 600 amino acids, from approximately 320 amino acids to approximately 550 amino acids, from approximately 320 amino acids to approximately 500 amino acids, from approximately 320 amino acids to approximately 480 amino acids, from approximately 320 amino acids to approximately 460 amino acids, from approximately 320 amino acids to approximately 440 amino acids, from approximately 320 amino acids to approximately 420 amino acids, from approximately 320 amino acids to approximately 400 amino acids, from approximately 320 amino acids to approximately 380 amino acids.Approximately 320 to approximately 360 amino acids, approximately 320 to approximately 340 amino acids, approximately 340 to approximately 3000 amino acids, approximately 340 to approximately 2500 amino acids, approximately 340 to approximately 2000 amino acids, approximately 340 to approximately 1500 amino acids, approximately 340 to approximately 1000 amino acids, approximately 340 to approximately 950 amino acids, approximately 340 to approximately 900 amino acids, approximately 340 to approximately 850 amino acids, approximately 340 to approximately 800 amino acids, approximately 340 to approximately 750 amino acids, approximately 340 to approximately 700 amino acids, approximately 340 amino acids... From approximately 650 amino acids, from approximately 340 amino acids to approximately 600 amino acids, from approximately 340 amino acids to approximately 550 amino acids, from approximately 340 amino acids to approximately 500 amino acids, from approximately 340 amino acids to approximately 480 amino acids, from approximately 340 amino acids to approximately 460 amino acids, from approximately 340 amino acids to approximately 440 amino acids, from approximately 340 amino acids to approximately 420 amino acids, from approximately 340 amino acids to approximately 400 amino acids, from approximately 340 amino acids to approximately 380 amino acids, from approximately 340 amino acids to approximately 360 amino acids, from approximately 360 amino acids to approximately 3000 amino acids, from approximately 360 amino acids to approximately 2500 amino acids, from approximately 360 amino acids to approximately 2000 amino acids. Approximately 360 amino acids to approximately 1500 amino acids, approximately 360 amino acids to approximately 1000 amino acids, approximately 360 amino acids to approximately 950 amino acids, approximately 360 amino acids to approximately 900 amino acids, approximately 360 amino acids to approximately 850 amino acids, approximately 360 amino acids to approximately 800 amino acids, approximately 360 amino acids to approximately 750 amino acids, approximately 360 amino acids to approximately 700 amino acids, approximately 360 amino acids to approximately 650 amino acids, approximately 360 amino acids to approximately 600 amino acids, approximately 360 amino acids to approximately 550 amino acids, approximately 360 amino acids to approximately 500 amino acids, approximately 360 amino acids to approximately 480 amino acids, approximately 360 amino acids to approximately... 460 amino acids, approximately 360 to approximately 440 amino acids, approximately 360 to approximately 420 amino acids, approximately 360 to approximately 400 amino acids, approximately 360 to approximately 380 amino acids, approximately 380 to approximately 3000 amino acids, approximately 380 to approximately 2500 amino acids, approximately 380 to approximately 2000 amino acids, approximately 380 to approximately 1500 amino acids, approximately 380 to approximately 1000 amino acids, approximately 380 to approximately 950 amino acids, approximately 380 to approximately 900 amino acids, approximately 380 to approximately 850 amino acids, approximately 380 to approximately 800 amino acids.Approximately 380 amino acids to approximately 750 amino acids, approximately 380 amino acids to approximately 700 amino acids, approximately 380 amino acids to approximately 650 amino acids, approximately 380 amino acids to approximately 600 amino acids, approximately 380 amino acids to approximately 550 amino acids, approximately 380 amino acids to approximately 500 amino acids, approximately 380 amino acids to approximately 480 amino acids, approximately 380 amino acids to approximately 460 amino acids, approximately 380 amino acids to approximately 440 amino acids, approximately 380 amino acids to approximately 420 amino acids, approximately 380 amino acids to approximately 400 amino acids, approximately 400 amino acids to approximately 3000 amino acids, approximately 400 amino acids to approximately 2500 amino acids, approximately 400 amino acids to approximately... 2000 amino acids, approximately 400 amino acids to approximately 1500 amino acids, approximately 400 amino acids to approximately 1000 amino acids, approximately 400 amino acids to approximately 950 amino acids, approximately 400 amino acids to approximately 900 amino acids, approximately 400 amino acids to approximately 850 amino acids, approximately 400 amino acids to approximately 800 amino acids, approximately 400 amino acids to approximately 750 amino acids, approximately 400 amino acids to approximately 700 amino acids, approximately 400 amino acids to approximately 650 amino acids, approximately 400 amino acids to approximately 600 amino acids, approximately 400 amino acids to approximately 550 amino acids, approximately 400 amino acids to approximately 500 amino acids, approximately 400 amino acids to approximately 480 amino acids, approximately 4 00 amino acids to approximately 460 amino acids, approximately 400 amino acids to approximately 440 amino acids, approximately 400 amino acids to approximately 420 amino acids, approximately 420 amino acids to approximately 3000 amino acids, approximately 420 amino acids to approximately 2500 amino acids, approximately 420 amino acids to approximately 2000 amino acids, approximately 420 amino acids to approximately 1500 amino acids, approximately 420 amino acids to approximately 1000 amino acids, approximately 420 amino acids to approximately 950 amino acids, approximately 420 amino acids to approximately 900 amino acids, approximately 420 amino acids to approximately 850 amino acids, approximately 420 amino acids to approximately 800 amino acids, approximately 420 amino acids to approximately 750 amino acids, approximately 420 amino acids to approximately 700 amino acids, approximately 420 to approximately 650 amino acids, approximately 420 to approximately 600 amino acids, approximately 420 to approximately 550 amino acids, approximately 420 to approximately 500 amino acids, approximately 420 to approximately 480 amino acids, approximately 420 to approximately 460 amino acids, approximately 420 to approximately 440 amino acids, approximately 440 to approximately 3000 amino acids, approximately 440 to approximately 2500 amino acids, approximately 440 to approximately 2000 amino acids, approximately 440 to approximately 1500 amino acids, approximately 440 to approximately 1000 amino acids, approximately 440 to approximately 950 amino acids.Approximately 440 amino acids to approximately 900 amino acids, approximately 440 amino acids to approximately 850 amino acids, approximately 440 amino acids to approximately 800 amino acids, approximately 440 amino acids to approximately 750 amino acids, approximately 440 amino acids to approximately 700 amino acids, approximately 440 amino acids to approximately 650 amino acids, approximately 440 amino acids to approximately 600 amino acids, approximately 440 amino acids to approximately 550 amino acids, approximately 440 amino acids to approximately 500 amino acids, approximately 440 amino acids to approximately 480 amino acids, approximately 440 amino acids to approximately 460 amino acids, approximately 460 amino acids to approximately 3000 amino acids, approximately 460 amino acids to approximately 2500 amino acids, approximately 460 amino acids to approximately... 2000 amino acids, approximately 460 amino acids to approximately 1500 amino acids, approximately 460 amino acids to approximately 1000 amino acids, approximately 460 amino acids to approximately 950 amino acids, approximately 460 amino acids to approximately 900 amino acids, approximately 460 amino acids to approximately 850 amino acids, approximately 460 amino acids to approximately 800 amino acids, approximately 460 amino acids to approximately 750 amino acids, approximately 460 amino acids to approximately 700 amino acids, approximately 460 amino acids to approximately 650 amino acids, approximately 460 amino acids to approximately 600 amino acids, approximately 460 amino acids to approximately 550 amino acids, approximately 460 amino acids to approximately 500 amino acids, approximately 460 amino acids to approximately 480 amino acids, approximately 4 From 80 amino acids to approximately 3000 amino acids, from approximately 480 amino acids to approximately 2500 amino acids, from approximately 480 amino acids to approximately 2000 amino acids, from approximately 480 amino acids to approximately 1500 amino acids, from approximately 480 amino acids to approximately 1000 amino acids, from approximately 480 amino acids to approximately 950 amino acids, from approximately 480 amino acids to approximately 900 amino acids, from approximately 480 amino acids to approximately 850 amino acids, from approximately 480 amino acids to approximately 800 amino acids, from approximately 480 amino acids to approximately 750 amino acids, from approximately 480 amino acids to approximately 700 amino acids, from approximately 480 amino acids to approximately 650 amino acids, from approximately 480 amino acids to approximately 600 amino acids, from approximately 480 amino acids to approximately... 550 amino acids, approximately 480 to approximately 500 amino acids, approximately 500 to approximately 3000 amino acids, approximately 500 to approximately 2500 amino acids, approximately 500 to approximately 2000 amino acids, approximately 500 to approximately 1500 amino acids, approximately 500 to approximately 1000 amino acids, approximately 500 to approximately 950 amino acids, approximately 500 to approximately 900 amino acids, approximately 500 to approximately 850 amino acids, approximately 500 to approximately 800 amino acids, approximately 500 to approximately 750 amino acids, approximately 500 to approximately 700 amino acids, approximately 500 to approximately 650 amino acids.Approximately 500 to approximately 600 amino acids, approximately 500 to approximately 550 amino acids, approximately 550 to approximately 3000 amino acids, approximately 550 to approximately 2500 amino acids, approximately 550 to approximately 2000 amino acids, approximately 550 to approximately 1500 amino acids, approximately 550 to approximately 1000 amino acids, approximately 550 to approximately 950 amino acids, approximately 550 to approximately 900 amino acids, approximately 550 to approximately 850 amino acids, approximately 550 to approximately 800 amino acids, approximately 550 to approximately 750 amino acids, approximately 550 to approximately 700 amino acids, approximately 550 to approximately... 650 amino acids, approximately 550 amino acids to approximately 600 amino acids, approximately 600 amino acids to approximately 3000 amino acids, approximately 600 amino acids to approximately 2500 amino acids, approximately 600 amino acids to approximately 2000 amino acids, approximately 600 amino acids to approximately 1500 amino acids, approximately 600 amino acids to approximately 1000 amino acids, approximately 600 amino acids to approximately 950 amino acids, approximately 600 amino acids to approximately 900 amino acids, approximately 600 amino acids to approximately 850 amino acids, approximately 600 amino acids to approximately 800 amino acids, approximately 600 amino acids to approximately 750 amino acids, approximately 600 amino acids to approximately 700 amino acids, approximately 600 amino acids to approximately 650 amino acids, approximately 6 From 50 amino acids to approximately 3000 amino acids, from approximately 650 amino acids to approximately 2500 amino acids, from approximately 650 amino acids to approximately 2000 amino acids, from approximately 650 amino acids to approximately 1500 amino acids, from approximately 650 amino acids to approximately 1000 amino acids, from approximately 650 amino acids to approximately 950 amino acids, from approximately 650 amino acids to approximately 900 amino acids, from approximately 650 amino acids to approximately 850 amino acids, from approximately 650 amino acids to approximately 800 amino acids, from approximately 650 amino acids to approximately 750 amino acids, from approximately 650 amino acids to approximately 700 amino acids, from approximately 700 amino acids to approximately 3000 amino acids, from approximately 700 amino acids to approximately 2500 amino acids, from approximately 700 amino acids to approximately... 2000 amino acids, approximately 700 to approximately 1500 amino acids, approximately 700 to approximately 1000 amino acids, approximately 700 to approximately 950 amino acids, approximately 700 to approximately 900 amino acids, approximately 700 to approximately 850 amino acids, approximately 700 to approximately 800 amino acids, approximately 700 to approximately 750 amino acids, approximately 750 to approximately 3000 amino acids, approximately 750 to approximately 2500 amino acids, approximately 750 to approximately 2000 amino acids, approximately 750 to approximately 1500 amino acids, approximately 750 to approximately 1000 amino acids, approximately 750 to approximately 950 amino acids.Approximately 750 to approximately 900 amino acids, approximately 750 to approximately 850 amino acids, approximately 750 to approximately 800 amino acids, approximately 800 to approximately 3000 amino acids, approximately 800 to approximately 2500 amino acids, approximately 800 to approximately 2000 amino acids, approximately 800 to approximately 1500 amino acids, approximately 800 to approximately 1000 amino acids, approximately 800 to approximately 950 amino acids, approximately 800 to approximately 900 amino acids. Approximately 800 to approximately 850 amino acids, approximately 850 to approximately 3000 amino acids, approximately 850 to approximately 2500 amino acids, approximately 850 to approximately 2000 amino acids, approximately 850 to approximately 1500 amino acids, approximately 850 to approximately 1000 amino acids, approximately 850 to approximately 950 amino acids, approximately 850 to approximately 900 amino acids, approximately 900 to approximately 3000 amino acids, approximately 900 to approximately 2500 amino acids Approximately 900 to approximately 2000 amino acids, approximately 900 to approximately 1500 amino acids, approximately 900 to approximately 1000 amino acids, approximately 900 to approximately 950 amino acids, approximately 950 to approximately 3000 amino acids, approximately 950 to approximately 2500 amino acids, approximately 950 to approximately 2000 amino acids, approximately 950 to approximately 1500 amino acids, approximately 950 to approximately 1000 amino acids, approximately 1000 to approximately 3000 amino acids. 0 amino acids, approximately 1000 amino acids to approximately 2500 amino acids, approximately 1000 amino acids to approximately 2000 amino acids, approximately 1000 amino acids to approximately 1500 amino acids, approximately 1500 amino acids to approximately 3000 amino acids, approximately 1500 amino acids to approximately 2500 amino acids, approximately 1500 amino acids to approximately 2000 amino acids, approximately 2000 amino acids to approximately 3000 amino acids, approximately 2000 amino acids to approximately 2500 amino acids, or approximately 2500 amino acids to approximately 3000 amino acids. The exemplary first multi-chain chimeric polypeptide provided herein is illustrated in [the relevant context]. Figure 1-4 In the middle. An exemplary second multi-chain chimeric polypeptide is depicted in the diagram. Figure 5-12 middle.

[0094] In some embodiments of any first and second multi-chain chimeric peptides described herein, a first target-binding domain (e.g., any first target-binding domain described herein) and a linker domain (e.g., any exemplary linker domain described herein) are directly adjacent to each other in the first chimeric peptide. In some embodiments of any first and second multi-chain chimeric peptides described herein, the first chimeric peptide further includes a linker sequence (any exemplary linker sequence described herein or known in the art) between the first target-binding domain (e.g., any exemplary first target-binding domain described herein) and the linker domain (e.g., any exemplary linker domain described herein) in the first chimeric peptide.

[0095] In some embodiments of any first and second multi-chain chimeric polypeptides described herein, a linker domain (e.g., any exemplary linker domain described herein) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of any first and second multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the linker domain (e.g., any exemplary linker domain described herein) and the first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) in the first chimeric polypeptide.

[0096] In some embodiments of any first and second multi-chain chimeric peptides described herein, the second domain of the affinity domain pair (e.g., any exemplary second domain of any exemplary affinity domain pair described herein) and the second target-binding domain (e.g., any exemplary second target-binding domain described herein) are directly adjacent to each other in the second chimeric peptide. In some embodiments of any first and second multi-chain chimeric peptides described herein, the second chimeric peptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the second domain of the affinity domain pair (e.g., any exemplary second domain of any exemplary affinity domain pair described herein) and the second target-binding domain (e.g., any exemplary second target-binding domain described herein) in the second chimeric peptide.

[0097] Organization Factor

[0098] Human tissue factor (CTF) is a 263-amino acid transmembrane protein containing three domains: (1) a 219-amino acid N-terminal extracellular domain (residues 1-219); (2) a 22-amino acid transmembrane domain (residues 220-242); and (3) a 21-amino acid C-terminal tail (residues 242-263) (UniProtKB identifier: P13726). The cytoplasmic tail is located in Ser... 253 and Ser 258 It contains two phosphorylation sites, and in Cys 245 It contains an S-palmitoylation site. No deletions or mutations of the cytoplasmic domain were found to affect the coagulation activity of tissue factor. Tissue factor is located in the intracellular domain of the protein in Cys... 245 It contains an S-palmitoylation site. Cys 245 Located at the amino acid terminus of the intracellular domain and close to the membrane surface, the transmembrane domain of tissue factor is composed of a single-span α-helix.

[0099] The extracellular domain of tissue factor, composed of two fibronectin type III domains, is linked to the transmembrane domain via a six-amino acid linker. This linker provides conformational flexibility to separate the tissue's extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module consists of two overlapping β-sheets, with the top fold containing three antiparallel β-chains and the bottom fold containing four β-chains. The β-chains are linked by β-loops between chains βA and βB, βC and βD, and βE and βF, which are conformationally conserved in both modules. Three short α-helical segments are present connecting the β-chains. A unique feature of tissue factor is the 17-amino acid β-hairpin between the β10 and β11 chains, which is not a common element in the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, a loop not present in the C-terminal domain and specific to tissue factor. This fibronectin type III domain structure is characteristic of immunoglobulin-like family protein folding and is conserved in a wide variety of extracellular proteins.

[0100] Once proenzyme FVII binds to tissue to form an active tissue factor-FVIIa complex, it can be rapidly converted to FVIIa through limited proteolysis. FVIIa circulating at a concentration of approximately 0.1 nM (1% of plasma FVII) can also bind directly to tissue factor. Allosteric interactions between tissue and FVIIa on the tissue factor-FVIIa complex significantly increase the enzymatic activity of FVIIa: the hydrolysis rate of small chromogenic peptide substrates increases by approximately 20 to 100 times, while the activation rate of natural macromolecular substrates FIX and FX increases by nearly a million times. Consistent with the allosteric activation of the FVIIa active site upon binding to tissue factor, the formation of the tissue-FVIIa complex on the phospholipid bilayer (i.e., exposure of phosphatidyl-L-serine on the membrane surface) activates FIX or FX at a rate that, in Ca... 2+ The dependence was increased by an additional 1,000-fold. The approximately million-fold overall increase in FX activation by the tissue-FVIIa-phospholipid complex relative to free FVIIa is a key regulator of the coagulation cascade.

[0101] FVII is a single-chain polypeptide of approximately 50 kDa, composed of 406 amino acid residues, containing an N-terminal γ-carboxyglutamate-rich domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII communicates with Ile- 154 -Arg 152 Specific proteolytic cleavage of the bond in the short linker region between EGF2 and the protease domain activates it to FVIIa. This cleavage induces the light and heavy chains to pass through Cys... 135 and Cys 262 The individual disulfide bonds hold them together. FVIIa is held together by its N-terminal GLA domain via Ca 2+ It binds to the phospholipid membrane in a dependent manner. The C-terminus of the GLA domain is adjacent to the aromatic stack and two EGF domains. The aromatic stack connects the GLA to the binding of a single Ca²⁺ molecule. 2+ The EGF1 domain of the ion. Occupying this Ca 2+ The binding site increases the amide degradation activity of FVIIa and tissue factor association. The catalytic triad is composed of His... 193 Asp 242 and Ser 344 Composition, and a single Ca within the FVIIa protease domain 2+ The binding of ions is crucial to their catalytic activity. FVII activates the proteolytic activity of FVIIa, releasing Ile. 153 The newly formed amino terminus is folded back and inserted into the activation bag, thereby interacting with Asp. 343The carboxylate forms a salt bridge to create an oxyanion pore. This salt bridge formation is crucial for FVIIa activity. However, upon proteolytic activation, the formation of oxyanion pores does not occur in free FVIIa. As a result, FVIIa cycles in its enzyme-as-an enzyme state, which plasma protease inhibitors do not readily recognize, thus allowing it to cycle with a half-life of approximately 90 minutes.

[0102] The tissue factor-mediated localization of the FVIIa active site above the membrane surface is important for FVIIa orientation toward homologous substrates. Free FVIIa, upon binding to the membrane, employs a stable extended structure, with its active site located approximately above the membrane surface. When FVIIa binds to tissue factor, the FVa active site relocates to a location closer to the membrane. This regulation helps the FVIIa catalytic triplet properly align with the target substrate cleavage site. Using FVIIa without the GLA domain, the active site has been shown to remain at a similar distance above the membrane, demonstrating that tissue factor can fully support the localization of the FVIIa active site even in the absence of FVIIa-membrane interactions. Additional data show that tissue factor supports full FVIIa proteolytic activity as long as the extracellular domain of tissue factor is somehow tethered to the membrane surface. However, elevating the FVIIa active site above the membrane surface requires more than [a certain amount of time / effort]. It will greatly reduce the ability of the tissue factor-FVIIa complex to activate FX, but will not reduce the amide decomposition activity of tissue factor-FVIIa.

[0103] Alanine scanning mutagenesis has been used to assess the role of specific amino acid side chains in the extracellular domain of tissue factor in their interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., Journal of Biol Chem 269(30):19399-19403, 1994). Alanine substitution identified a limited number of residue sites where alanine substitution resulted in a 5- to 10-fold decrease in affinity for FVIIa binding. Most of these residue side chains were found to be well exposed to the solvent in the crystal structure and consistent with interactions with macromolecular ligands. The FVIIa ligand binding sites were located over a broad region at the boundary between the two modules. In module C, the Arg residue located on the prominent BC ring... 135 and Phe 140 Provides independent contact with FVIIa. Leu 133 Located at the base of the finger-like structure, and squeezed into the crack between the two modules. This is provided by Lys 20 Thr 60 Asp58 and Ile 22 The continuity of the main clusters of important binding residues. Thr 60 It is only partially exposed to the solvent and may play a localized structural role rather than having significant contact with the ligand. The binding site extends to the concave surface of the intermodal corner, which involves Glu. 24 and Gln 110 And may involve more distant residues Val 207 The binding region extends from Asp58 to Lys 48 Lys 46 、Gln 37 Asp 44 and Trp 45 The resulting convex surface region. Trp 45 and Asp 44 It does not interact independently with FVIIa, which suggests that Trp 45 The mutation effect at this location may reflect the effect of this side chain on neighboring Asp. 44 and Gln 37 The structural importance of localized side chain stacking. The interaction region further contains two surface-exposed aromatic residues, Phe 76 and Tyr 78 It forms part of the hydrophobic cluster in the N module.

[0104] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as receptors activated by certain proteases. Mutation analysis has identified numerous residues that, when mutated, support full FVIIa amideolytic activity against small peptide substrates but lack the ability to support activation against macromolecular substrates (i.e., FVII, FIX, and FX) (Ruf et al., Journal of Biochemistry 267(31):22206-22210, 1992; Ruf et al., Journal of Biochemistry 267(9):6375-6381, 1992; Huang et al., Journal of Biochemistry 271(36):21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region at residues 159-165 and residues in or near this flexible loop have been shown to be crucial for the proteolytic activity of the tissue factor-FVIIa complex. This defines the extra-substrate binding site region of the proposed tissue factor, which is located far from the active site of FVIIa. Substitution of glycine residues with the slightly larger alanine residue significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility provided by glycine is crucial for the loop at residues 159-165 used for macromolecular substrate recognition by tissue factor.

[0105] It also proved the residue Lys 165 and Lys 166 This is important for substrate recognition and binding. Mutations of any of these residues to alanine result in a significant reduction in the function of tissue factor cofactors. In most tissue factor-FVIIa structures, Lys 165 and Lys 166 Back to back, Lys 165 Pointing to FVIIa, while Lys 166 Pointing to the region outside the substrate binding site in the crystal structure. Lys of FVIIa 165 and Gla 35 The proposed salt bridge formation between them would support the idea that the interaction between tissue factor and the GLA domain of FVIIa regulates substrate recognition. These results suggest that the C-terminal portion of the extracellular domain of tissue factor interacts directly with the GLA domains of FIX and FX, and possibly the neighboring EGF1 domain, and that the presence of the FVIIa GLA domain can directly or indirectly modulate these interactions.

[0106] Exemplary adapter domains and IgG1 antibody constructs

[0107] In some instances of any of the methods, compositions, or kits described herein, the linker domain in the second multi-chain chimeric polypeptide may be or contains any antigen recognized by a homologous IgG1 antibody (e.g., a monoclonal antibody). IgG1 antibodies differ from other IgG classes of antibodies in their constant regions, specifically the hinge region and the upper CH2 domain (see, for example, Vadarsson et al., “IgG Subclasses and Allotypes: From Structure to Effector Functions”, Frontiers in Immunology, 5, Chapter 520, 2014). Human IgG1 is the only known IgG subclass that binds to and activates human CD16a signaling.

[0108] In the second multi-chain chimeric polypeptide provided herein, any of a variety of adapter domains may be used. In some embodiments, the adapter domain is recognized by an antibody (e.g., an IgG1 antibody) or an antibody fragment. In some embodiments, the adapter domain is an antibody κ light chain recognized by a homologous anti-κ light chain IgG1 antibody. In some embodiments, the adapter domain is an antibody λ light chain recognized by a homologous anti-λ light chain human IgG1 antibody. Multiple κ and λ light chains are known in the art (see, for example, 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 adapter domain is or includes a human polypeptide (e.g., the human κ light chain or the human λ light chain of the antibody) recognized by a homologous human IgG1 antibody (e.g., a monoclonal antibody). In some embodiments, the homologous human IgG1 antibody comprises at least two antigen-binding domains, and each antigen-binding domain specifically binds to the adapter domain. In some embodiments, the homologous human IgG1 antibody comprises at least two antigen-binding domains, and only a subset of the antigen-binding domains (e.g., one antigen-binding domain) specifically binds to the adapter domain in the second multi-chain chimeric polypeptide. In some embodiments, the adapter domain may be any soluble tissue factor domain described herein.

[0109] In some embodiments of any of the methods, compositions, and kits described herein, the IgG1 antibody construct may 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 may be an antibody or antibody fragment comprising an IgG1 Fc region (e.g., a human IgG1 Fc region) and specifically binding to a linker domain in a second multi-chain chimeric polypeptide. As is known in the art, the IgG1 Fc region binds to CD16a (FcRγIII) (e.g., human CD16a) and induces its intracellular signaling. In some embodiments, the IgG1 antibody construct may be a single-chain or multi-chain polypeptide comprising an Fc region capable of specifically binding to CD16a (FcRγIII) (e.g., human CD16a) and inducing its intracellular signaling in natural killer cells (e.g., human natural killer cells), and specifically binding to a linker domain. In some embodiments, the IgG1 antibody construct may be a single-chain or multi-chain polypeptide containing an Fc region and specifically binding to a linker domain, the Fc region containing a sequence having at least 80% identity (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) with wild-type IgG1 Fc domains (e.g., wild-type human IgG1 Fc domains, e.g., SEQ ID NO:1) and being capable of specifically binding to CD16a (FcRγIII) (e.g., human CD16a) and being capable of inducing its intracellular signaling in natural killer cells (e.g., human natural killer cells). In some embodiments of any of the methods, compositions, or kits described herein, the IgG1 antibody construct may be an antibody or antibody fragment that specifically binds to a linker domain and includes a non-IgG1 Fc region (e.g., an IgG2, IgG3, or IgG4 Fc region) that has been 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) so that the non-IgG1 Fc region can bind to CD16a (FcRγIII) (e.g., human CD16a) and induce its intracellular signaling in natural killer cells (e.g., human natural killer cells).In some embodiments of any of the methods, compositions, or kits described herein, the IgG1 antibody construct specifically binds to a linker domain in a second multi-chain chimeric polypeptide and includes a non-human Fc region (e.g., an Fc region derived from a non-human antibody) that has been 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) so that the non-human Fc region can bind to human CD16a (human FcRγIII) and induce its intracellular signaling in natural killer cells (e.g., human natural killer cells).

[0110] Wild-type human IgG1 Fc region (SEQ ID NO:1)

[0111] PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0112] Wild-type human IgG2 Fc region (SEQ ID NO:2)

[0113] APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0114] Wild-type human IgG3 Fc region (SEQ ID NO:3)

[0115] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

[0116] Wild-type human IgG4 Fc region (SEQ ID NO:4)

[0117] APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0118] In some embodiments, the total length of the connector domain can be 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, and from about 20 amino acids to about 160 amino acids. Approximately 155 amino acids, approximately 20 amino acids to approximately 150 amino acids, approximately 20 amino acids to approximately 145 amino acids, approximately 20 amino acids to approximately 140 amino acids, approximately 20 amino acids to approximately 135 amino acids, approximately 20 amino acids to approximately 130 amino acids, approximately 20 amino acids to approximately 125 amino acids, approximately 20 amino acids to approximately 120 amino acids, approximately 20 amino acids to approximately 115 amino acids, approximately 20 amino acids to approximately 110 amino acids, approximately 20 amino acids to approximately 105 amino acids, approximately 20 amino acids to approximately 100 amino acids, approximately 20 amino acids to approximately 95 amino acids, approximately 20 amino acids to approximately 90 amino acids, approximately 20 amino acids to approximately 85 amino acids, approximately 20 amino acids... From approximately 80 amino acids, from approximately 20 amino acids to approximately 75 amino acids, from approximately 20 amino acids to approximately 70 amino acids, from approximately 20 amino acids to approximately 60 amino acids, from approximately 20 amino acids to approximately 50 amino acids, from approximately 20 amino acids to approximately 40 amino acids, from approximately 20 amino acids to approximately 30 amino acids, from approximately 30 amino acids to approximately 220 amino acids, from approximately 30 amino acids to approximately 215 amino acids, from approximately 30 amino acids to approximately 210 amino acids, from approximately 30 amino acids to approximately 205 amino acids, from approximately 30 amino acids to approximately 200 amino acids, from approximately 30 amino acids to approximately 195 amino acids, from approximately 30 amino acids to approximately 190 amino acids, from approximately 30 amino acids to approximately 185 amino acids, from approximately 30 amino acids... From approximately 180 amino acids, from approximately 30 amino acids to approximately 175 amino acids, from approximately 30 amino acids to approximately 170 amino acids, from approximately 30 amino acids to approximately 165 amino acids, from approximately 30 amino acids to approximately 160 amino acids, from approximately 30 amino acids to approximately 155 amino acids, from approximately 30 amino acids to approximately 150 amino acids, from approximately 30 amino acids to approximately 145 amino acids, from approximately 30 amino acids to approximately 140 amino acids, from approximately 30 amino acids to approximately 135 amino acids, from approximately 30 amino acids to approximately 130 amino acids, from approximately 30 amino acids to approximately 125 amino acids, from approximately 30 amino acids to approximately 120 amino acids, from approximately 30 amino acids to approximately 115 amino acids, from approximately 30 amino acids to approximately 110 amino acids.Approximately 30 to approximately 105 amino acids, approximately 30 to approximately 100 amino acids, approximately 30 to approximately 95 amino acids, approximately 30 to approximately 90 amino acids, approximately 30 to approximately 85 amino acids, approximately 30 to approximately 80 amino acids, approximately 30 to approximately 75 amino acids, approximately 30 to approximately 70 amino acids, approximately 30 to approximately 60 amino acids, approximately 30 to approximately 50 amino acids, approximately 30 to approximately 40 amino acids, approximately 40 to approximately 220 amino acids, approximately 40 to approximately 215 amino acids, approximately 40 to approximately 210 amino acids, approximately 40 to approximately 205 amino acids. From approximately 40 amino acids to approximately 200 amino acids, from approximately 40 amino acids to approximately 195 amino acids, from approximately 40 amino acids to approximately 190 amino acids, from approximately 40 amino acids to approximately 185 amino acids, from approximately 40 amino acids to approximately 180 amino acids, from approximately 40 amino acids to approximately 175 amino acids, from approximately 40 amino acids to approximately 170 amino acids, from approximately 40 amino acids to approximately 165 amino acids, from approximately 40 amino acids to approximately 160 amino acids, from approximately 40 amino acids to approximately 155 amino acids, from approximately 40 amino acids to approximately 150 amino acids, from approximately 40 amino acids to approximately 145 amino acids, from approximately 40 amino acids to approximately 140 amino acids, from approximately 40 amino acids to approximately 135 amino acids, from approximately 40 amino acids to approximately... 130 amino acids, approximately 40 amino acids to approximately 125 amino acids, approximately 40 amino acids to approximately 120 amino acids, approximately 40 amino acids to approximately 115 amino acids, approximately 40 amino acids to approximately 110 amino acids, approximately 40 amino acids to approximately 105 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 95 amino acids, approximately 40 amino acids to approximately 90 amino acids, approximately 40 amino acids to approximately 85 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 75 amino acids, approximately 40 amino acids to approximately 70 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 40 amino acids to approximately 50 amino acids, approximately 50 amino acids to... Approximately 220 amino acids, approximately 50 to approximately 215 amino acids, approximately 50 to approximately 210 amino acids, approximately 50 to approximately 205 amino acids, approximately 50 to approximately 200 amino acids, approximately 50 to approximately 195 amino acids, approximately 50 to approximately 190 amino acids, approximately 50 to approximately 185 amino acids, approximately 50 to approximately 180 amino acids, approximately 50 to approximately 175 amino acids, approximately 50 to approximately 170 amino acids, approximately 50 to approximately 165 amino acids, approximately 50 to approximately 160 amino acids, approximately 50 to approximately 155 amino acids, approximately 50 to approximately 150 amino acids.Approximately 50 amino acids to approximately 145 amino acids, approximately 50 amino acids to approximately 140 amino acids, approximately 50 amino acids to approximately 135 amino acids, approximately 50 amino acids to approximately 130 amino acids, approximately 50 amino acids to approximately 125 amino acids, approximately 50 amino acids to approximately 120 amino acids, approximately 50 amino acids to approximately 115 amino acids, approximately 50 amino acids to approximately 110 amino acids, approximately 50 amino acids to approximately 105 amino acids, approximately 50 amino acids to approximately 100 amino acids, approximately 50 amino acids to approximately 95 amino acids, approximately 50 amino acids to approximately 90 amino acids, approximately 50 amino acids to approximately 85 amino acids, approximately 50 amino acids to approximately 80 amino acids, approximately 50 amino acids to approximately 75 amino acids. Amino acids, approximately 50 to approximately 70 amino acids, approximately 50 to approximately 60 amino acids, approximately 60 to approximately 220 amino acids, approximately 60 to approximately 215 amino acids, approximately 60 to approximately 210 amino acids, approximately 60 to approximately 205 amino acids, approximately 60 to approximately 200 amino acids, approximately 60 to approximately 195 amino acids, approximately 60 to approximately 190 amino acids, approximately 60 to approximately 185 amino acids, approximately 60 to approximately 180 amino acids, approximately 60 to approximately 175 amino acids, approximately 60 to approximately 170 amino acids, approximately 60 to approximately 165 amino acids, approximately 60 to approximately... 160 amino acids, approximately 60 to approximately 155 amino acids, approximately 60 to approximately 150 amino acids, approximately 60 to approximately 145 amino acids, approximately 60 to approximately 140 amino acids, approximately 60 to approximately 135 amino acids, approximately 60 to approximately 130 amino acids, approximately 60 to approximately 125 amino acids, approximately 60 to approximately 120 amino acids, approximately 60 to approximately 115 amino acids, approximately 60 to approximately 110 amino acids, approximately 60 to approximately 105 amino acids, approximately 60 to approximately 100 amino acids, approximately 60 to approximately 95 amino acids, approximately 60 to approximately 90 amino acids, approximately 60 From approximately 85 amino acids, from approximately 60 amino acids to approximately 80 amino acids, from approximately 60 amino acids to approximately 75 amino acids, from approximately 60 amino acids to approximately 70 amino acids, from approximately 70 amino acids to approximately 220 amino acids, from approximately 70 amino acids to approximately 215 amino acids, from approximately 70 amino acids to approximately 210 amino acids, from approximately 70 amino acids to approximately 205 amino acids, from approximately 70 amino acids to approximately 200 amino acids, from approximately 70 amino acids to approximately 195 amino acids, from approximately 70 amino acids to approximately 190 amino acids, from approximately 70 amino acids to approximately 185 amino acids, from approximately 70 amino acids to approximately 180 amino acids, from approximately 70 amino acids to approximately 175 amino acids, from approximately 70 amino acids to approximately 170 amino acids.From approximately 70 amino acids to approximately 165 amino acids, from approximately 70 amino acids to approximately 160 amino acids, from approximately 70 amino acids to approximately 155 amino acids, from approximately 70 amino acids to approximately 150 amino acids, from approximately 70 amino acids to approximately 145 amino acids, from approximately 70 amino acids to approximately 140 amino acids, from approximately 70 amino acids to approximately 135 amino acids, from approximately 70 amino acids to approximately 130 amino acids, from approximately 70 amino acids to approximately 125 amino acids, from approximately 70 amino acids to approximately 120 amino acids, from approximately 70 amino acids to approximately 115 amino acids, from approximately 70 amino acids to approximately 110 amino acids, from approximately 70 amino acids to approximately 105 amino acids, from approximately 70 amino acids to approximately 100 amino acids, from approximately 70 amino acids to approximately 95 amino acids. 70 to 90 amino acids, approximately 70 to 85 amino acids, approximately 70 to 80 amino acids, approximately 80 to 220 amino acids, approximately 80 to 215 amino acids, approximately 80 to 210 amino acids, approximately 80 to 205 amino acids, approximately 80 to 200 amino acids, approximately 80 to 195 amino acids, approximately 80 to 185 amino acids, approximately 80 to 180 amino acids, approximately 80 to 175 amino acids, approximately 80 to 170 amino acids, approximately 80 to approximately... 165 amino acids, approximately 80 amino acids to approximately 160 amino acids, approximately 80 amino acids to approximately 155 amino acids, approximately 80 amino acids to approximately 150 amino acids, approximately 80 amino acids to approximately 145 amino acids, approximately 80 amino acids to approximately 140 amino acids, approximately 80 amino acids to approximately 135 amino acids, approximately 80 amino acids to approximately 130 amino acids, approximately 80 amino acids to approximately 125 amino acids, approximately 80 amino acids to approximately 120 amino acids, approximately 80 amino acids to approximately 115 amino acids, approximately 80 amino acids to approximately 110 amino acids, approximately 80 amino acids to approximately 105 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 80 amino acids to approximately 95 amino acids, approximately 80 From approximately 90 amino acids, from approximately 90 amino acids to approximately 220 amino acids, from approximately 90 amino acids to approximately 215 amino acids, from approximately 90 amino acids to approximately 210 amino acids, from approximately 90 amino acids to approximately 205 amino acids, from approximately 90 amino acids to approximately 200 amino acids, from approximately 90 amino acids to approximately 195 amino acids, from approximately 90 amino acids to approximately 190 amino acids, from approximately 90 amino acids to approximately 185 amino acids, from approximately 90 amino acids to approximately 180 amino acids, from approximately 90 amino acids to approximately 175 amino acids, from approximately 90 amino acids to approximately 170 amino acids, from approximately 90 amino acids to approximately 165 amino acids, from approximately 90 amino acids to approximately 160 amino acids, from approximately 90 amino acids to approximately 155 amino acids.Approximately 90 to approximately 150 amino acids, approximately 90 to approximately 145 amino acids, approximately 90 to approximately 140 amino acids, approximately 90 to approximately 135 amino acids, approximately 90 to approximately 130 amino acids, approximately 90 to approximately 125 amino acids, approximately 90 to approximately 120 amino acids, approximately 90 to approximately 115 amino acids, approximately 90 to approximately 110 amino acids, approximately 90 to approximately 105 amino acids, approximately 90 to approximately 100 amino acids, approximately 100 to approximately 220 amino acids, approximately 100 to approximately 215 amino acids, approximately 100 to approximately 210 amino acids, approximately 100 amino acids From approximately 205 amino acids, from approximately 100 amino acids to approximately 200 amino acids, from approximately 100 amino acids to approximately 195 amino acids, from approximately 100 amino acids to approximately 190 amino acids, from approximately 100 amino acids to approximately 185 amino acids, from approximately 100 amino acids to approximately 180 amino acids, from approximately 100 amino acids to approximately 175 amino acids, from approximately 100 amino acids to approximately 170 amino acids, from approximately 100 amino acids to approximately 165 amino acids, from approximately 100 amino acids to approximately 160 amino acids, from approximately 100 amino acids to approximately 155 amino acids, from approximately 100 amino acids to approximately 150 amino acids, from approximately 100 amino acids to approximately 145 amino acids, from approximately 100 amino acids to approximately 140 amino acids, from approximately 100 amino acids... From approximately 135 amino acids, from approximately 100 amino acids to approximately 130 amino acids, from approximately 100 amino acids to approximately 125 amino acids, from approximately 100 amino acids to approximately 120 amino acids, from approximately 100 amino acids to approximately 115 amino acids, from approximately 100 amino acids to approximately 110 amino acids, from approximately 110 amino acids to approximately 220 amino acids, from approximately 110 amino acids to approximately 215 amino acids, from approximately 110 amino acids to approximately 210 amino acids, from approximately 110 amino acids to approximately 205 amino acids, from approximately 110 amino acids to approximately 200 amino acids, from approximately 110 amino acids to approximately 195 amino acids, from approximately 110 amino acids to approximately 190 amino acids, from approximately 110 amino acids to approximately 185 amino acids, from approximately 1 From 10 amino acids to approximately 180 amino acids, from approximately 110 amino acids to approximately 175 amino acids, from approximately 110 amino acids to approximately 170 amino acids, from approximately 110 amino acids to approximately 165 amino acids, from approximately 110 amino acids to approximately 160 amino acids, from approximately 110 amino acids to approximately 155 amino acids, from approximately 110 amino acids to approximately 150 amino acids, from approximately 110 amino acids to approximately 145 amino acids, from approximately 110 amino acids to approximately 140 amino acids, from approximately 110 amino acids to approximately 135 amino acids, from approximately 110 amino acids to approximately 130 amino acids, from approximately 110 amino acids to approximately 125 amino acids, from approximately 110 amino acids to approximately 120 amino acids, from approximately 110 amino acids to approximately 115 amino acids.From approximately 115 amino acids to approximately 220 amino acids, from approximately 115 amino acids to approximately 215 amino acids, from approximately 115 amino acids to approximately 210 amino acids, from approximately 115 amino acids to approximately 205 amino acids, from approximately 115 amino acids to approximately 200 amino acids, from approximately 115 amino acids to approximately 195 amino acids, from approximately 115 amino acids to approximately 190 amino acids, from approximately 115 amino acids to approximately 185 amino acids, from approximately 115 amino acids to approximately 180 amino acids, from approximately 115 amino acids to approximately 175 amino acids, from approximately 115 amino acids to approximately 170 amino acids, from approximately 115 amino acids to approximately 165 amino acids, from approximately 115 amino acids to approximately 160 amino acids, from approximately 115 amino acids to approximately 155 amino acids. 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to Approximately 190 amino acids, approximately 120 to approximately 185 amino acids, approximately 120 to approximately 180 amino acids, approximately 120 to approximately 175 amino acids, approximately 120 to approximately 170 amino acids, approximately 120 to approximately 165 amino acids, approximately 120 to approximately 160 amino acids, approximately 120 to approximately 155 amino acids, approximately 120 to approximately 150 amino acids, approximately 120 to approximately 145 amino acids, approximately 120 to approximately 140 amino acids, approximately 120 to approximately 135 amino acids, approximately 120 to approximately 130 amino acids, approximately 120 to approximately 125 amino acids, approximately 125 From approximately 125 amino acids to approximately 215 amino acids, from approximately 125 amino acids to approximately 210 amino acids, from approximately 125 amino acids to approximately 205 amino acids, from approximately 125 amino acids to approximately 200 amino acids, from approximately 125 amino acids to approximately 195 amino acids, from approximately 125 amino acids to approximately 190 amino acids, from approximately 125 amino acids to approximately 185 amino acids, from approximately 125 amino acids to approximately 180 amino acids, from approximately 125 amino acids to approximately 175 amino acids, from approximately 125 amino acids to approximately 170 amino acids, from approximately 125 amino acids to approximately 165 amino acids, from approximately 125 amino acids to approximately 160 amino acids, from approximately 125 amino acids to approximately 155 amino acids.From approximately 125 amino acids to approximately 150 amino acids, from approximately 125 amino acids to approximately 145 amino acids, from approximately 125 amino acids to approximately 140 amino acids, from approximately 125 amino acids to approximately 135 amino acids, from approximately 125 amino acids to approximately 130 amino acids, from approximately 130 amino acids to approximately 220 amino acids, from approximately 130 amino acids to approximately 215 amino acids, from approximately 130 amino acids to approximately 210 amino acids, from approximately 130 amino acids to approximately 205 amino acids, from approximately 130 amino acids to approximately 200 amino acids, from approximately 130 amino acids to approximately 195 amino acids, from approximately 130 amino acids to approximately 190 amino acids, from approximately 130 amino acids to approximately 185 amino acids, from approximately 130 amino acids to approximately 180 amino acids. 130 amino acids to about 175 amino acids, approximately 130 amino acids to about 170 amino acids, approximately 130 amino acids to about 165 amino acids, approximately 130 amino acids to about 160 amino acids, approximately 130 amino acids to about 155 amino acids, approximately 130 amino acids to about 150 amino acids, approximately 130 amino acids to about 145 amino acids, approximately 130 amino acids to about 140 amino acids, approximately 130 amino acids to about 135 amino acids, approximately 135 amino acids to about 220 amino acids, approximately 135 amino acids to about 215 amino acids, approximately 135 amino acids to about 210 amino acids, approximately 135 amino acids to about 205 amino acids, approximately 135 amino acids to Approximately 200 amino acids, approximately 135 to approximately 195 amino acids, approximately 135 to approximately 190 amino acids, approximately 135 to approximately 185 amino acids, approximately 135 to approximately 180 amino acids, approximately 135 to approximately 175 amino acids, approximately 135 to approximately 170 amino acids, approximately 135 to approximately 165 amino acids, approximately 135 to approximately 160 amino acids, approximately 135 to approximately 155 amino acids, approximately 135 to approximately 150 amino acids, approximately 135 to approximately 145 amino acids, approximately 135 to approximately 140 amino acids, approximately 140 to approximately 220 amino acids, approximately 140 From approximately 140 amino acids to approximately 215 amino acids, from approximately 140 amino acids to approximately 210 amino acids, from approximately 140 amino acids to approximately 205 amino acids, from approximately 140 amino acids to approximately 200 amino acids, from approximately 140 amino acids to approximately 195 amino acids, from approximately 140 amino acids to approximately 190 amino acids, from approximately 140 amino acids to approximately 185 amino acids, from approximately 140 amino acids to approximately 180 amino acids, from approximately 140 amino acids to approximately 175 amino acids, from approximately 140 amino acids to approximately 170 amino acids, from approximately 140 amino acids to approximately 165 amino acids, from approximately 140 amino acids to approximately 160 amino acids, from approximately 140 amino acids to approximately 155 amino acids, from approximately 140 amino acids to approximately 150 amino acids.From approximately 140 amino acids to approximately 145 amino acids, from approximately 145 amino acids to approximately 220 amino acids, from approximately 145 amino acids to approximately 215 amino acids, from approximately 145 amino acids to approximately 210 amino acids, from approximately 145 amino acids to approximately 205 amino acids, from approximately 145 amino acids to approximately 200 amino acids, from approximately 145 amino acids to approximately 195 amino acids, from approximately 145 amino acids to approximately 190 amino acids, from approximately 145 amino acids to approximately 185 amino acids, from approximately 145 amino acids to approximately 180 amino acids, from approximately 145 amino acids to approximately 175 amino acids, from approximately 145 amino acids to approximately 170 amino acids, from approximately 145 amino acids to approximately 165 amino acids, from approximately 145 amino acids to approximately 160 amino acids. 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to Approximately 165 amino acids, approximately 150 to approximately 160 amino acids, approximately 150 to approximately 155 amino acids, approximately 155 to approximately 220 amino acids, approximately 155 to approximately 215 amino acids, approximately 155 to approximately 210 amino acids, approximately 155 to approximately 205 amino acids, approximately 155 to approximately 200 amino acids, approximately 155 to approximately 195 amino acids, approximately 155 to approximately 190 amino acids, approximately 155 to approximately 185 amino acids, approximately 155 to approximately 180 amino acids, approximately 155 to approximately 175 amino acids, approximately 155 to approximately 170 amino acids, approximately 155 From approximately 165 amino acids, from approximately 155 amino acids to approximately 160 amino acids, from approximately 160 amino acids to approximately 220 amino acids, from approximately 160 amino acids to approximately 215 amino acids, from approximately 160 amino acids to approximately 210 amino acids, from approximately 160 amino acids to approximately 205 amino acids, from approximately 160 amino acids to approximately 200 amino acids, from approximately 160 amino acids to approximately 195 amino acids, from approximately 160 amino acids to approximately 190 amino acids, from approximately 160 amino acids to approximately 185 amino acids, from approximately 160 amino acids to approximately 180 amino acids, from approximately 160 amino acids to approximately 175 amino acids, from approximately 160 amino acids to approximately 170 amino acids, from approximately 160 amino acids to approximately 165 amino acids.From approximately 165 amino acids to approximately 220 amino acids, from approximately 165 amino acids to approximately 215 amino acids, from approximately 165 amino acids to approximately 210 amino acids, from approximately 165 amino acids to approximately 205 amino acids, from approximately 165 amino acids to approximately 200 amino acids, from approximately 165 amino acids to approximately 195 amino acids, from approximately 165 amino acids to approximately 190 amino acids, from approximately 165 amino acids to approximately 185 amino acids, from approximately 165 amino acids to approximately 180 amino acids, from approximately 165 amino acids to approximately 175 amino acids, from approximately 165 amino acids to approximately 170 amino acids, from approximately 170 amino acids to approximately 220 amino acids, from approximately 170 amino acids to approximately 215 amino acids, from approximately 170 amino acids to approximately 210... 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to Approximately 190 amino acids, approximately 175 amino acids to approximately 185 amino acids, approximately 175 amino acids to approximately 180 amino acids, approximately 180 amino acids to approximately 220 amino acids, approximately 180 amino acids to approximately 215 amino acids, approximately 180 amino acids to approximately 210 amino acids, approximately 180 amino acids to approximately 205 amino acids, approximately 180 amino acids to approximately 200 amino acids, approximately 180 amino acids to approximately 195 amino acids, approximately 180 amino acids to approximately 190 amino acids, approximately 180 amino acids to approximately 185 amino acids, approximately 185 amino acids to approximately 220 amino acids, approximately 185 amino acids to approximately 215 amino acids, approximately 185 amino acids to approximately 210 amino acids, approximately 185 From approximately 185 amino acids to approximately 205 amino acids, from approximately 185 amino acids to approximately 200 amino acids, from approximately 185 amino acids to approximately 195 amino acids, from approximately 185 amino acids to approximately 190 amino acids, from approximately 190 amino acids to approximately 220 amino acids, from approximately 190 amino acids to approximately 215 amino acids, from approximately 190 amino acids to approximately 205 amino acids, from approximately 190 amino acids to approximately 200 amino acids, from approximately 190 amino acids to approximately 195 amino acids, from approximately 195 amino acids to approximately 220 amino acids, from approximately 195 amino acids to approximately 215 amino acids, from approximately 195 amino acids to approximately 210 amino acids, from approximately 195 amino acids to approximately 205 amino acids.Approximately 195 to approximately 200 amino acids, approximately 200 to approximately 220 amino acids, approximately 200 to approximately 215 amino acids, approximately 200 to approximately 210 amino acids, approximately 200 to approximately 205 amino acids, approximately 205 to approximately 220 amino acids, approximately 205 to approximately 215 amino acids, approximately 205 to approximately 210 amino acids, approximately 210 to approximately 220 amino acids, approximately 210 to approximately 215 amino acids, or approximately 215 to approximately 220 amino acids.

[0119] Soluble tissue factor domain

[0120] In some examples of any of the methods, compositions, or kits described herein, the linker domain may be a soluble tissue factor domain. In some embodiments, the soluble tissue factor domain may be a wild-type tissue factor peptide lacking a signal sequence, a transmembrane domain, and an intracellular domain. In some examples, the soluble tissue factor domain may be a tissue factor mutant, wherein the wild-type tissue factor peptide lacks a signal sequence, a transmembrane domain, and an intracellular domain, and has been further modified at selected amino acids. In some examples, the soluble tissue factor domain may be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain may be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, mouse soluble tissue factor domains, rat soluble tissue factor domains, and mutant soluble tissue factor domains are shown below.

[0121] Exemplary soluble human tissue factor domain (SEQ ID NO:5)

[0122] SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0123] An exemplary nucleic acid encoding a soluble human tissue factor domain (SEQ ID NO:6)

[0124] AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

[0125] Exemplary soluble mouse tissue factor domain (SEQ ID NO:7)

[0126] agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge

[0127] Exemplary soluble rat tissue factor domain (SEQ ID NO:8)

[0128] Agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

[0129] Exemplary mutant soluble human tissue factor domain (SEQ ID NO:9)

[0130] SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0131] Exemplary mutant soluble human tissue factor domain (SEQ ID NO:10)

[0132] SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0133] In some embodiments, the soluble tissue factor domain may comprise a sequence having at least 70% identity, at least 72% identity, at least 74% identity, at least 76% identity, at least 78% identity, at least 80% identity, at least 82% identity, at least 84% identity, at least 86% identity, at least 88% identity, at least 90% identity, at least 92% identity, at least 94% identity, at least 96% identity, at least 98% identity, at least 99% identity, or 100% identity with SEQ ID NO:5, 7, 8, 9, or 100%. In some embodiments, the soluble tissue factor domain may comprise the sequence SEQ ID NO:5, 7, 8, 9, or 10, wherein one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) have been removed from its N-terminus and / or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) have been removed from its C-terminus.

[0134] As those skilled in the art will understand, mutations in conserved amino acids across different mammalian species are more likely to reduce protein activity and / or structural stability, while mutations in non-conserved amino acids across different mammalian species are less likely to reduce protein activity and / or structural stability.

[0135] In some instances, the soluble tissue factor domain cannot bind factor VIIa. In some instances, the soluble tissue factor domain does not convert inactive factor X into factor Xa. In some embodiments, the single-chain chimeric polypeptide does not stimulate blood clotting in mammals.

[0136] In some instances, the soluble tissue factor domain may be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble rat tissue factor domain.

[0137] In some instances, the soluble tissue factor domain does not contain one or more of the following (e.g., two, three, four, five, six, or seven): lysine at amino acid position 20 corresponding to amino acid position 2 ...

[0138] In some embodiments, the soluble tissue factor domain may be encoded by a nucleic acid comprising a sequence having at least 70% identity, at least 72% identity, at least 74% identity, at least 76% identity, at least 78% identity, at least 80% identity, at least 82% identity, at least 84% identity, at least 86% identity, at least 88% identity, at least 90% identity, at least 92% identity, at least 94% identity, at least 96% identity, at least 98% identity, at least 99% identity, or 100% identity with SEQ ID NO:6.

[0139] In some embodiments, the total length of the soluble tissue factor domain can be 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, or about 20 amino acids. From approximately 155 amino acids, from approximately 20 amino acids to approximately 150 amino acids, from approximately 20 amino acids to approximately 145 amino acids, from approximately 20 amino acids to approximately 140 amino acids, from approximately 20 amino acids to approximately 135 amino acids, from approximately 20 amino acids to approximately 130 amino acids, from approximately 20 amino acids to approximately 125 amino acids, from approximately 20 amino acids to approximately 120 amino acids, from approximately 20 amino acids to approximately 115 amino acids, from approximately 20 amino acids to approximately 110 amino acids, from approximately 20 amino acids to approximately 105 amino acids, from approximately 20 amino acids to approximately 100 amino acids, from approximately 20 amino acids to approximately 95 amino acids, from approximately 20 amino acids to approximately 90 amino acids, from approximately 20 amino acids to approximately 85 amino acids, from approximately 2 0 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 From approximately 180 amino acids, from approximately 30 amino acids to approximately 175 amino acids, from approximately 30 amino acids to approximately 170 amino acids, from approximately 30 amino acids to approximately 165 amino acids, from approximately 30 amino acids to approximately 160 amino acids, from approximately 30 amino acids to approximately 155 amino acids, from approximately 30 amino acids to approximately 150 amino acids, from approximately 30 amino acids to approximately 145 amino acids, from approximately 30 amino acids to approximately 140 amino acids, from approximately 30 amino acids to approximately 135 amino acids, from approximately 30 amino acids to approximately 130 amino acids, from approximately 30 amino acids to approximately 125 amino acids, from approximately 30 amino acids to approximately 120 amino acids, from approximately 30 amino acids to approximately 115 amino acids, from approximately 30 amino acids to approximately 110 amino acids.Approximately 30 to approximately 105 amino acids, approximately 30 to approximately 100 amino acids, approximately 30 to approximately 95 amino acids, approximately 30 to approximately 90 amino acids, approximately 30 to approximately 85 amino acids, approximately 30 to approximately 80 amino acids, approximately 30 to approximately 75 amino acids, approximately 30 to approximately 70 amino acids, approximately 30 to approximately 60 amino acids, approximately 30 to approximately 50 amino acids, approximately 30 to approximately 40 amino acids, approximately 40 to approximately 220 amino acids, approximately 40 to approximately 215 amino acids, approximately 40 to approximately 210 amino acids, approximately 40 to approximately 205 amino acids. From approximately 40 amino acids to approximately 200 amino acids, from approximately 40 amino acids to approximately 195 amino acids, from approximately 40 amino acids to approximately 190 amino acids, from approximately 40 amino acids to approximately 185 amino acids, from approximately 40 amino acids to approximately 180 amino acids, from approximately 40 amino acids to approximately 175 amino acids, from approximately 40 amino acids to approximately 170 amino acids, from approximately 40 amino acids to approximately 165 amino acids, from approximately 40 amino acids to approximately 160 amino acids, from approximately 40 amino acids to approximately 155 amino acids, from approximately 40 amino acids to approximately 150 amino acids, from approximately 40 amino acids to approximately 145 amino acids, from approximately 40 amino acids to approximately 140 amino acids, from approximately 40 amino acids to approximately 135 amino acids, from approximately 40 amino acids to approximately... 130 amino acids, approximately 40 amino acids to approximately 125 amino acids, approximately 40 amino acids to approximately 120 amino acids, approximately 40 amino acids to approximately 115 amino acids, approximately 40 amino acids to approximately 110 amino acids, approximately 40 amino acids to approximately 105 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 95 amino acids, approximately 40 amino acids to approximately 90 amino acids, approximately 40 amino acids to approximately 85 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 75 amino acids, approximately 40 amino acids to approximately 70 amino acids, approximately 40 amino acids to approximately 60 amino acids, approximately 40 amino acids to approximately 50 amino acids, approximately 50 amino acids to... Approximately 220 amino acids, approximately 50 to approximately 215 amino acids, approximately 50 to approximately 210 amino acids, approximately 50 to approximately 205 amino acids, approximately 50 to approximately 200 amino acids, approximately 50 to approximately 195 amino acids, approximately 50 to approximately 190 amino acids, approximately 50 to approximately 185 amino acids, approximately 50 to approximately 180 amino acids, approximately 50 to approximately 175 amino acids, approximately 50 to approximately 170 amino acids, approximately 50 to approximately 165 amino acids, approximately 50 to approximately 160 amino acids, approximately 50 to approximately 155 amino acids, approximately 50 to approximately 150 amino acids.Approximately 50 amino acids to approximately 145 amino acids, approximately 50 amino acids to approximately 140 amino acids, approximately 50 amino acids to approximately 135 amino acids, approximately 50 amino acids to approximately 130 amino acids, approximately 50 amino acids to approximately 125 amino acids, approximately 50 amino acids to approximately 120 amino acids, approximately 50 amino acids to approximately 115 amino acids, approximately 50 amino acids to approximately 110 amino acids, approximately 50 amino acids to approximately 105 amino acids, approximately 50 amino acids to approximately 100 amino acids, approximately 50 amino acids to approximately 95 amino acids, approximately 50 amino acids to approximately 90 amino acids, approximately 50 amino acids to approximately 85 amino acids, approximately 50 amino acids to approximately 80 amino acids, approximately 50 amino acids to approximately 75 amino acids. Amino acids, approximately 50 to approximately 70 amino acids, approximately 50 to approximately 60 amino acids, approximately 60 to approximately 220 amino acids, approximately 60 to approximately 215 amino acids, approximately 60 to approximately 210 amino acids, approximately 60 to approximately 205 amino acids, approximately 60 to approximately 200 amino acids, approximately 60 to approximately 195 amino acids, approximately 60 to approximately 190 amino acids, approximately 60 to approximately 185 amino acids, approximately 60 to approximately 180 amino acids, approximately 60 to approximately 175 amino acids, approximately 60 to approximately 170 amino acids, approximately 60 to approximately 165 amino acids, approximately 60 to approximately... 160 amino acids, approximately 60 to approximately 155 amino acids, approximately 60 to approximately 150 amino acids, approximately 60 to approximately 145 amino acids, approximately 60 to approximately 140 amino acids, approximately 60 to approximately 135 amino acids, approximately 60 to approximately 130 amino acids, approximately 60 to approximately 125 amino acids, approximately 60 to approximately 120 amino acids, approximately 60 to approximately 115 amino acids, approximately 60 to approximately 110 amino acids, approximately 60 to approximately 105 amino acids, approximately 60 to approximately 100 amino acids, approximately 60 to approximately 95 amino acids, approximately 60 to approximately 90 amino acids, approximately 60 From approximately 85 amino acids, from approximately 60 amino acids to approximately 80 amino acids, from approximately 60 amino acids to approximately 75 amino acids, from approximately 60 amino acids to approximately 70 amino acids, from approximately 70 amino acids to approximately 220 amino acids, from approximately 70 amino acids to approximately 215 amino acids, from approximately 70 amino acids to approximately 210 amino acids, from approximately 70 amino acids to approximately 205 amino acids, from approximately 70 amino acids to approximately 200 amino acids, from approximately 70 amino acids to approximately 195 amino acids, from approximately 70 amino acids to approximately 190 amino acids, from approximately 70 amino acids to approximately 185 amino acids, from approximately 70 amino acids to approximately 180 amino acids, from approximately 70 amino acids to approximately 175 amino acids, from approximately 70 amino acids to approximately 170 amino acids.From approximately 70 amino acids to approximately 165 amino acids, from approximately 70 amino acids to approximately 160 amino acids, from approximately 70 amino acids to approximately 155 amino acids, from approximately 70 amino acids to approximately 150 amino acids, from approximately 70 amino acids to approximately 145 amino acids, from approximately 70 amino acids to approximately 140 amino acids, from approximately 70 amino acids to approximately 135 amino acids, from approximately 70 amino acids to approximately 130 amino acids, from approximately 70 amino acids to approximately 125 amino acids, from approximately 70 amino acids to approximately 120 amino acids, from approximately 70 amino acids to approximately 115 amino acids, from approximately 70 amino acids to approximately 110 amino acids, from approximately 70 amino acids to approximately 105 amino acids, from approximately 70 amino acids to approximately 100 amino acids, from approximately 70 amino acids to approximately 95 amino acids. 70 to 90 amino acids, approximately 70 to 85 amino acids, approximately 70 to 80 amino acids, approximately 80 to 220 amino acids, approximately 80 to 215 amino acids, approximately 80 to 210 amino acids, approximately 80 to 205 amino acids, approximately 80 to 200 amino acids, approximately 80 to 195 amino acids, approximately 80 to 185 amino acids, approximately 80 to 180 amino acids, approximately 80 to 175 amino acids, approximately 80 to 170 amino acids, approximately 80 to approximately... 165 amino acids, approximately 80 amino acids to approximately 160 amino acids, approximately 80 amino acids to approximately 155 amino acids, approximately 80 amino acids to approximately 150 amino acids, approximately 80 amino acids to approximately 145 amino acids, approximately 80 amino acids to approximately 140 amino acids, approximately 80 amino acids to approximately 135 amino acids, approximately 80 amino acids to approximately 130 amino acids, approximately 80 amino acids to approximately 125 amino acids, approximately 80 amino acids to approximately 120 amino acids, approximately 80 amino acids to approximately 115 amino acids, approximately 80 amino acids to approximately 110 amino acids, approximately 80 amino acids to approximately 105 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 80 amino acids to approximately 95 amino acids, approximately 80 From approximately 90 amino acids, from approximately 90 amino acids to approximately 220 amino acids, from approximately 90 amino acids to approximately 215 amino acids, from approximately 90 amino acids to approximately 210 amino acids, from approximately 90 amino acids to approximately 205 amino acids, from approximately 90 amino acids to approximately 200 amino acids, from approximately 90 amino acids to approximately 195 amino acids, from approximately 90 amino acids to approximately 190 amino acids, from approximately 90 amino acids to approximately 185 amino acids, from approximately 90 amino acids to approximately 180 amino acids, from approximately 90 amino acids to approximately 175 amino acids, from approximately 90 amino acids to approximately 170 amino acids, from approximately 90 amino acids to approximately 165 amino acids, from approximately 90 amino acids to approximately 160 amino acids, from approximately 90 amino acids to approximately 155 amino acids.Approximately 90 to approximately 150 amino acids, approximately 90 to approximately 145 amino acids, approximately 90 to approximately 140 amino acids, approximately 90 to approximately 135 amino acids, approximately 90 to approximately 130 amino acids, approximately 90 to approximately 125 amino acids, approximately 90 to approximately 120 amino acids, approximately 90 to approximately 115 amino acids, approximately 90 to approximately 110 amino acids, approximately 90 to approximately 105 amino acids, approximately 90 to approximately 100 amino acids, approximately 100 to approximately 220 amino acids, approximately 100 to approximately 215 amino acids, approximately 100 to approximately 210 amino acids, approximately 100 amino acids From approximately 205 amino acids, from approximately 100 amino acids to approximately 200 amino acids, from approximately 100 amino acids to approximately 195 amino acids, from approximately 100 amino acids to approximately 190 amino acids, from approximately 100 amino acids to approximately 185 amino acids, from approximately 100 amino acids to approximately 180 amino acids, from approximately 100 amino acids to approximately 175 amino acids, from approximately 100 amino acids to approximately 170 amino acids, from approximately 100 amino acids to approximately 165 amino acids, from approximately 100 amino acids to approximately 160 amino acids, from approximately 100 amino acids to approximately 155 amino acids, from approximately 100 amino acids to approximately 150 amino acids, from approximately 100 amino acids to approximately 145 amino acids, from approximately 100 amino acids to approximately 140 amino acids, from approximately 100 amino acids... From approximately 135 amino acids, from approximately 100 amino acids to approximately 130 amino acids, from approximately 100 amino acids to approximately 125 amino acids, from approximately 100 amino acids to approximately 120 amino acids, from approximately 100 amino acids to approximately 115 amino acids, from approximately 100 amino acids to approximately 110 amino acids, from approximately 110 amino acids to approximately 220 amino acids, from approximately 110 amino acids to approximately 215 amino acids, from approximately 110 amino acids to approximately 210 amino acids, from approximately 110 amino acids to approximately 205 amino acids, from approximately 110 amino acids to approximately 200 amino acids, from approximately 110 amino acids to approximately 195 amino acids, from approximately 110 amino acids to approximately 190 amino acids, from approximately 110 amino acids to approximately 185 amino acids, from approximately 1 From 10 amino acids to approximately 180 amino acids, from approximately 110 amino acids to approximately 175 amino acids, from approximately 110 amino acids to approximately 170 amino acids, from approximately 110 amino acids to approximately 165 amino acids, from approximately 110 amino acids to approximately 160 amino acids, from approximately 110 amino acids to approximately 155 amino acids, from approximately 110 amino acids to approximately 150 amino acids, from approximately 110 amino acids to approximately 145 amino acids, from approximately 110 amino acids to approximately 140 amino acids, from approximately 110 amino acids to approximately 135 amino acids, from approximately 110 amino acids to approximately 130 amino acids, from approximately 110 amino acids to approximately 125 amino acids, from approximately 110 amino acids to approximately 120 amino acids, from approximately 110 amino acids to approximately 115 amino acids.From approximately 115 amino acids to approximately 220 amino acids, from approximately 115 amino acids to approximately 215 amino acids, from approximately 115 amino acids to approximately 210 amino acids, from approximately 115 amino acids to approximately 205 amino acids, from approximately 115 amino acids to approximately 200 amino acids, from approximately 115 amino acids to approximately 195 amino acids, from approximately 115 amino acids to approximately 190 amino acids, from approximately 115 amino acids to approximately 185 amino acids, from approximately 115 amino acids to approximately 180 amino acids, from approximately 115 amino acids to approximately 175 amino acids, from approximately 115 amino acids to approximately 170 amino acids, from approximately 115 amino acids to approximately 165 amino acids, from approximately 115 amino acids to approximately 160 amino acids, from approximately 115 amino acids to approximately 155 amino acids. 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to Approximately 190 amino acids, approximately 120 to approximately 185 amino acids, approximately 120 to approximately 180 amino acids, approximately 120 to approximately 175 amino acids, approximately 120 to approximately 170 amino acids, approximately 120 to approximately 165 amino acids, approximately 120 to approximately 160 amino acids, approximately 120 to approximately 155 amino acids, approximately 120 to approximately 150 amino acids, approximately 120 to approximately 145 amino acids, approximately 120 to approximately 140 amino acids, approximately 120 to approximately 135 amino acids, approximately 120 to approximately 130 amino acids, approximately 120 to approximately 125 amino acids, approximately 125 From approximately 125 amino acids to approximately 215 amino acids, from approximately 125 amino acids to approximately 210 amino acids, from approximately 125 amino acids to approximately 205 amino acids, from approximately 125 amino acids to approximately 200 amino acids, from approximately 125 amino acids to approximately 195 amino acids, from approximately 125 amino acids to approximately 190 amino acids, from approximately 125 amino acids to approximately 185 amino acids, from approximately 125 amino acids to approximately 180 amino acids, from approximately 125 amino acids to approximately 175 amino acids, from approximately 125 amino acids to approximately 170 amino acids, from approximately 125 amino acids to approximately 165 amino acids, from approximately 125 amino acids to approximately 160 amino acids, from approximately 125 amino acids to approximately 155 amino acids.From approximately 125 amino acids to approximately 150 amino acids, from approximately 125 amino acids to approximately 145 amino acids, from approximately 125 amino acids to approximately 140 amino acids, from approximately 125 amino acids to approximately 135 amino acids, from approximately 125 amino acids to approximately 130 amino acids, from approximately 130 amino acids to approximately 220 amino acids, from approximately 130 amino acids to approximately 215 amino acids, from approximately 130 amino acids to approximately 210 amino acids, from approximately 130 amino acids to approximately 205 amino acids, from approximately 130 amino acids to approximately 200 amino acids, from approximately 130 amino acids to approximately 195 amino acids, from approximately 130 amino acids to approximately 190 amino acids, from approximately 130 amino acids to approximately 185 amino acids, from approximately 130 amino acids to approximately 180 amino acids. 130 amino acids to about 175 amino acids, approximately 130 amino acids to about 170 amino acids, approximately 130 amino acids to about 165 amino acids, approximately 130 amino acids to about 160 amino acids, approximately 130 amino acids to about 155 amino acids, approximately 130 amino acids to about 150 amino acids, approximately 130 amino acids to about 145 amino acids, approximately 130 amino acids to about 140 amino acids, approximately 130 amino acids to about 135 amino acids, approximately 135 amino acids to about 220 amino acids, approximately 135 amino acids to about 215 amino acids, approximately 135 amino acids to about 210 amino acids, approximately 135 amino acids to about 205 amino acids, approximately 135 amino acids to Approximately 200 amino acids, approximately 135 to approximately 195 amino acids, approximately 135 to approximately 190 amino acids, approximately 135 to approximately 185 amino acids, approximately 135 to approximately 180 amino acids, approximately 135 to approximately 175 amino acids, approximately 135 to approximately 170 amino acids, approximately 135 to approximately 165 amino acids, approximately 135 to approximately 160 amino acids, approximately 135 to approximately 155 amino acids, approximately 135 to approximately 150 amino acids, approximately 135 to approximately 145 amino acids, approximately 135 to approximately 140 amino acids, approximately 140 to approximately 220 amino acids, approximately 140 From approximately 140 amino acids to approximately 215 amino acids, from approximately 140 amino acids to approximately 210 amino acids, from approximately 140 amino acids to approximately 205 amino acids, from approximately 140 amino acids to approximately 200 amino acids, from approximately 140 amino acids to approximately 195 amino acids, from approximately 140 amino acids to approximately 190 amino acids, from approximately 140 amino acids to approximately 185 amino acids, from approximately 140 amino acids to approximately 180 amino acids, from approximately 140 amino acids to approximately 175 amino acids, from approximately 140 amino acids to approximately 170 amino acids, from approximately 140 amino acids to approximately 165 amino acids, from approximately 140 amino acids to approximately 160 amino acids, from approximately 140 amino acids to approximately 155 amino acids, from approximately 140 amino acids to approximately 150 amino acids.From approximately 140 amino acids to approximately 145 amino acids, from approximately 145 amino acids to approximately 220 amino acids, from approximately 145 amino acids to approximately 215 amino acids, from approximately 145 amino acids to approximately 210 amino acids, from approximately 145 amino acids to approximately 205 amino acids, from approximately 145 amino acids to approximately 200 amino acids, from approximately 145 amino acids to approximately 195 amino acids, from approximately 145 amino acids to approximately 190 amino acids, from approximately 145 amino acids to approximately 185 amino acids, from approximately 145 amino acids to approximately 180 amino acids, from approximately 145 amino acids to approximately 175 amino acids, from approximately 145 amino acids to approximately 170 amino acids, from approximately 145 amino acids to approximately 165 amino acids, from approximately 145 amino acids to approximately 160 amino acids. 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to Approximately 165 amino acids, approximately 150 to approximately 160 amino acids, approximately 150 to approximately 155 amino acids, approximately 155 to approximately 220 amino acids, approximately 155 to approximately 215 amino acids, approximately 155 to approximately 210 amino acids, approximately 155 to approximately 205 amino acids, approximately 155 to approximately 200 amino acids, approximately 155 to approximately 195 amino acids, approximately 155 to approximately 190 amino acids, approximately 155 to approximately 185 amino acids, approximately 155 to approximately 180 amino acids, approximately 155 to approximately 175 amino acids, approximately 155 to approximately 170 amino acids, approximately 155 From approximately 165 amino acids, from approximately 155 amino acids to approximately 160 amino acids, from approximately 160 amino acids to approximately 220 amino acids, from approximately 160 amino acids to approximately 215 amino acids, from approximately 160 amino acids to approximately 210 amino acids, from approximately 160 amino acids to approximately 205 amino acids, from approximately 160 amino acids to approximately 200 amino acids, from approximately 160 amino acids to approximately 195 amino acids, from approximately 160 amino acids to approximately 190 amino acids, from approximately 160 amino acids to approximately 185 amino acids, from approximately 160 amino acids to approximately 180 amino acids, from approximately 160 amino acids to approximately 175 amino acids, from approximately 160 amino acids to approximately 170 amino acids, from approximately 160 amino acids to approximately 165 amino acids.From approximately 165 amino acids to approximately 220 amino acids, from approximately 165 amino acids to approximately 215 amino acids, from approximately 165 amino acids to approximately 210 amino acids, from approximately 165 amino acids to approximately 205 amino acids, from approximately 165 amino acids to approximately 200 amino acids, from approximately 165 amino acids to approximately 195 amino acids, from approximately 165 amino acids to approximately 190 amino acids, from approximately 165 amino acids to approximately 185 amino acids, from approximately 165 amino acids to approximately 180 amino acids, from approximately 165 amino acids to approximately 175 amino acids, from approximately 165 amino acids to approximately 170 amino acids, from approximately 170 amino acids to approximately 220 amino acids, from approximately 170 amino acids to approximately 215 amino acids, from approximately 170 amino acids to approximately 210... 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to Approximately 190 amino acids, approximately 175 amino acids to approximately 185 amino acids, approximately 175 amino acids to approximately 180 amino acids, approximately 180 amino acids to approximately 220 amino acids, approximately 180 amino acids to approximately 215 amino acids, approximately 180 amino acids to approximately 210 amino acids, approximately 180 amino acids to approximately 205 amino acids, approximately 180 amino acids to approximately 200 amino acids, approximately 180 amino acids to approximately 195 amino acids, approximately 180 amino acids to approximately 190 amino acids, approximately 180 amino acids to approximately 185 amino acids, approximately 185 amino acids to approximately 220 amino acids, approximately 185 amino acids to approximately 215 amino acids, approximately 185 amino acids to approximately 210 amino acids, approximately 185 From approximately 185 amino acids to approximately 205 amino acids, from approximately 185 amino acids to approximately 200 amino acids, from approximately 185 amino acids to approximately 195 amino acids, from approximately 185 amino acids to approximately 190 amino acids, from approximately 190 amino acids to approximately 220 amino acids, from approximately 190 amino acids to approximately 215 amino acids, from approximately 190 amino acids to approximately 205 amino acids, from approximately 190 amino acids to approximately 200 amino acids, from approximately 190 amino acids to approximately 195 amino acids, from approximately 195 amino acids to approximately 220 amino acids, from approximately 195 amino acids to approximately 215 amino acids, from approximately 195 amino acids to approximately 210 amino acids, from approximately 195 amino acids to approximately 205 amino acids.Approximately 195 to approximately 200 amino acids, approximately 200 to approximately 220 amino acids, approximately 200 to approximately 215 amino acids, approximately 200 to approximately 210 amino acids, approximately 200 to approximately 205 amino acids, approximately 205 to approximately 220 amino acids, approximately 205 to approximately 215 amino acids, approximately 205 to approximately 210 amino acids, approximately 210 to approximately 220 amino acids, approximately 210 to approximately 215 amino acids, or approximately 215 to approximately 220 amino acids.

[0140] Connector sequence

[0141] In some embodiments, the adapter sequence may be a flexible adapter sequence. Non-limiting examples of adapter sequences that may be used are described in the following literature: Klein et al., Protein Engineering, Design & Selection, Vol. 27, No. 10, pp. 325-330, 2014; Priyanka et al., Protein Sci., Feb. 2013; 22(2):153–167. In some instances, the adapter sequence is a synthetic adapter sequence.

[0142] In some embodiments of any multi-chain chimeric polypeptide described herein, the first and / or second polypeptide may comprise one, two, three, four, five, six, seven, eight, nine, or ten adapter sequences (e.g., the same or different adapter sequences, such as any exemplary adapter sequences described herein or known in the art).

[0143] In some embodiments, the total length of the linker sequence can be from 1 amino acid to about 100 amino acids, from 1 amino acid to about 90 amino acids, from 1 amino acid to about 80 amino acids, from 1 amino acid to about 70 amino acids, from 1 amino acid to about 60 amino acids, from 1 amino acid to about 50 amino acids, from 1 amino acid to about 45 amino acids, from 1 amino acid to about 40 amino acids, from 1 amino acid to about 35 amino acids, from 1 amino acid to about 30 amino acids, from 1 amino acid to about 25 amino acids, from 1 amino acid to about 24 amino acids, from 1 amino acid to about 22 amino acids, from 1 amino acid to about 20 amino acids, from 1 amino acid to about 18 amino acids, from 1 amino acid to about 16 amino acids, or from 1 amino acid... From approximately 14 amino acids, 1 amino acid to approximately 12 amino acids, 1 amino acid to approximately 10 amino acids, 1 amino acid to approximately 8 amino acids, 1 amino acid to approximately 6 amino acids, 1 amino acid to approximately 4 amino acids, approximately 2 amino acids to approximately 100 amino acids, approximately 2 amino acids to approximately 90 amino acids, approximately 2 amino acids to approximately 80 amino acids, approximately 2 amino acids to approximately 70 amino acids, approximately 2 amino acids to approximately 60 amino acids, approximately 2 amino acids to approximately 50 amino acids, approximately 2 amino acids to approximately 45 amino acids, approximately 2 amino acids to approximately 40 amino acids, approximately 2 amino acids to approximately 35 amino acids, approximately 2 amino acids to approximately 30 amino acids, approximately 2 amino acids to approximately 25 amino acids, approximately 2 amino acids to approximately... 24 amino acids, approximately 2 amino acids to approximately 22 amino acids, approximately 2 amino acids to approximately 20 amino acids, approximately 2 amino acids to approximately 18 amino acids, approximately 2 amino acids to approximately 16 amino acids, approximately 2 amino acids to approximately 14 amino acids, approximately 2 amino acids to approximately 12 amino acids, approximately 2 amino acids to approximately 10 amino acids, approximately 2 amino acids to approximately 8 amino acids, approximately 2 amino acids to approximately 6 amino acids, approximately 2 amino acids to approximately 4 amino acids, approximately 4 amino acids to approximately 100 amino acids, approximately 4 amino acids to approximately 90 amino acids, approximately 4 amino acids to approximately 80 amino acids, approximately 4 amino acids to approximately 70 amino acids, approximately 4 amino acids to approximately 60 amino acids, approximately 4 amino acids to approximately 50 amino acids, approximately 4 amino acids From approximately 45 amino acids, from approximately 4 amino acids to approximately 40 amino acids, from approximately 4 amino acids to approximately 35 amino acids, from approximately 4 amino acids to approximately 30 amino acids, from approximately 4 amino acids to approximately 25 amino acids, from approximately 4 amino acids to approximately 24 amino acids, from approximately 4 amino acids to approximately 22 amino acids, from approximately 4 amino acids to approximately 20 amino acids, from approximately 4 amino acids to approximately 18 amino acids, from approximately 4 amino acids to approximately 16 amino acids, from approximately 4 amino acids to approximately 14 amino acids, from approximately 4 amino acids to approximately 12 amino acids, from approximately 4 amino acids to approximately 10 amino acids, from approximately 4 amino acids to approximately 8 amino acids, from approximately 4 amino acids to approximately 6 amino acids, from approximately 6 amino acids to approximately 100 amino acids, from approximately 6 amino acids to approximately 90 amino acids.From approximately 6 amino acids to approximately 80 amino acids, from approximately 6 amino acids to approximately 70 amino acids, from approximately 6 amino acids to approximately 60 amino acids, from approximately 6 amino acids to approximately 50 amino acids, from approximately 6 amino acids to approximately 45 amino acids, from approximately 6 amino acids to approximately 40 amino acids, from approximately 6 amino acids to approximately 35 amino acids, from approximately 6 amino acids to approximately 30 amino acids, from approximately 6 amino acids to approximately 25 amino acids, from approximately 6 amino acids to approximately 24 amino acids, from approximately 6 amino acids to approximately 22 amino acids, from approximately 6 amino acids to approximately 20 amino acids, from approximately 6 amino acids to approximately 18 amino acids, from approximately 6 amino acids to approximately 16 amino acids, from approximately 6 amino acids to approximately 14 amino acids, from approximately 6 amino acids to approximately 12 amino acids, from approximately 6 amino acids to... Approximately 10 amino acids, approximately 6 amino acids to approximately 8 amino acids, approximately 8 amino acids to approximately 100 amino acids, approximately 8 amino acids to approximately 90 amino acids, approximately 8 amino acids to approximately 80 amino acids, approximately 8 amino acids to approximately 70 amino acids, approximately 8 amino acids to approximately 60 amino acids, approximately 8 amino acids to approximately 50 amino acids, approximately 8 amino acids to approximately 45 amino acids, approximately 8 amino acids to approximately 40 amino acids, approximately 8 amino acids to approximately 35 amino acids, approximately 8 amino acids to approximately 30 amino acids, approximately 8 amino acids to approximately 25 amino acids, approximately 8 amino acids to approximately 24 amino acids, approximately 8 amino acids to approximately 22 amino acids, approximately 8 amino acids to approximately 20 amino acids, approximately 8 amino acids to approximately 18 amino acids. Approximately 8 to approximately 16 amino acids, approximately 8 to approximately 14 amino acids, approximately 8 to approximately 12 amino acids, approximately 8 to approximately 10 amino acids, approximately 10 to approximately 100 amino acids, approximately 10 to approximately 90 amino acids, approximately 10 to approximately 80 amino acids, approximately 10 to approximately 70 amino acids, approximately 10 to approximately 60 amino acids, approximately 10 to approximately 50 amino acids, approximately 10 to approximately 45 amino acids, approximately 10 to approximately 40 amino acids, approximately 10 to approximately 35 amino acids, approximately 10 to approximately 30 amino acids, approximately 10 to approximately 25 amino acids, approximately 10 to approximately... 24 amino acids, approximately 10 to approximately 22 amino acids, approximately 10 to approximately 20 amino acids, approximately 10 to approximately 18 amino acids, approximately 10 to approximately 16 amino acids, approximately 10 to approximately 14 amino acids, approximately 10 to approximately 12 amino acids, approximately 12 to approximately 100 amino acids, approximately 12 to approximately 90 amino acids, approximately 12 to approximately 80 amino acids, approximately 12 to approximately 70 amino acids, approximately 12 to approximately 60 amino acids, approximately 12 to approximately 50 amino acids, approximately 12 to approximately 45 amino acids, approximately 12 to approximately 40 amino acids, approximately 12 to approximately 35 amino acids.From approximately 12 amino acids to approximately 30 amino acids, from approximately 12 amino acids to approximately 25 amino acids, from approximately 12 amino acids to approximately 24 amino acids, from approximately 12 amino acids to approximately 22 amino acids, from approximately 12 amino acids to approximately 20 amino acids, from approximately 12 amino acids to approximately 18 amino acids, from approximately 12 amino acids to approximately 16 amino acids, from approximately 12 amino acids to approximately 14 amino acids, from approximately 14 amino acids to approximately 100 amino acids, from approximately 14 amino acids to approximately 90 amino acids, from approximately 14 amino acids to approximately 80 amino acids, from approximately 14 amino acids to approximately 70 amino acids, from approximately 14 amino acids to approximately 60 amino acids, from approximately 14 amino acids to approximately 50 amino acids, from approximately 14 amino acids to approximately 45 amino acids, from approximately 14 amino acids to... Approximately 40 amino acids, approximately 14 to approximately 35 amino acids, approximately 14 to approximately 30 amino acids, approximately 14 to approximately 25 amino acids, approximately 14 to approximately 24 amino acids, approximately 14 to approximately 22 amino acids, approximately 14 to approximately 20 amino acids, approximately 14 to approximately 18 amino acids, approximately 14 to approximately 16 amino acids, approximately 16 to approximately 100 amino acids, approximately 16 to approximately 90 amino acids, approximately 16 to approximately 80 amino acids, approximately 16 to approximately 70 amino acids, approximately 16 to approximately 60 amino acids, approximately 16 to approximately 50 amino acids, approximately 16 to approximately 45 amino acids. Approximately 16 amino acids to approximately 40 amino acids, approximately 16 amino acids to approximately 35 amino acids, approximately 16 amino acids to approximately 30 amino acids, approximately 16 amino acids to approximately 25 amino acids, approximately 16 amino acids to approximately 24 amino acids, approximately 16 amino acids to approximately 22 amino acids, approximately 16 amino acids to approximately 20 amino acids, approximately 16 amino acids to approximately 18 amino acids, approximately 18 amino acids to approximately 100 amino acids, approximately 18 amino acids to approximately 90 amino acids, approximately 18 amino acids to approximately 80 amino acids, approximately 18 amino acids to approximately 70 amino acids, approximately 18 amino acids to approximately 60 amino acids, approximately 18 amino acids to approximately 50 amino acids, approximately 18 amino acids to approximately 45 amino acids, approximately 18 amino acids to Approximately 40 amino acids, approximately 18 to approximately 35 amino acids, approximately 18 to approximately 30 amino acids, approximately 18 to approximately 25 amino acids, approximately 18 to approximately 24 amino acids, approximately 18 to approximately 22 amino acids, approximately 18 to approximately 20 amino acids, approximately 20 to approximately 100 amino acids, approximately 20 to approximately 90 amino acids, approximately 20 to approximately 80 amino acids, approximately 20 to approximately 70 amino acids, approximately 20 to approximately 60 amino acids, approximately 20 to approximately 50 amino acids, approximately 20 to approximately 45 amino acids, approximately 20 to approximately 40 amino acids, approximately 20 to approximately 35 amino acids.Approximately 20 to approximately 30 amino acids, approximately 20 to approximately 25 amino acids, approximately 20 to approximately 24 amino acids, approximately 20 to approximately 22 amino acids, approximately 22 to approximately 100 amino acids, approximately 22 to approximately 90 amino acids, approximately 22 to approximately 80 amino acids, approximately 22 to approximately 70 amino acids, approximately 22 to approximately 60 amino acids, approximately 22 to approximately 50 amino acids, approximately 22 to approximately 45 amino acids, approximately 22 to approximately 40 amino acids, approximately 22 to approximately 35 amino acids, approximately 22 to approximately 30 amino acids, approximately 22 to approximately 25 amino acids, approximately 22 to approximately... 24 amino acids, approximately 25 amino acids to approximately 100 amino acids, approximately 25 amino acids to approximately 90 amino acids, approximately 25 amino acids to approximately 80 amino acids, approximately 25 amino acids to approximately 70 amino acids, approximately 25 amino acids to approximately 60 amino acids, approximately 25 amino acids to approximately 50 amino acids, approximately 25 amino acids to approximately 45 amino acids, approximately 25 amino acids to approximately 40 amino acids, approximately 25 amino acids to approximately 35 amino acids, approximately 25 amino acids to approximately 30 amino acids, approximately 30 amino acids to approximately 100 amino acids, approximately 30 amino acids to approximately 90 amino acids, approximately 30 amino acids to approximately 80 amino acids, approximately 30 amino acids to approximately 70 amino acids, approximately 30 amino acids to approximately 60 amino acids, approximately 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, approximately 40 to approximately 60 amino acids, approximately 40 to approximately 50 amino acids, approximately 40 to approximately 45 amino acids, approximately 45 to approximately 100 amino acids, approximately 45 to approximately 90 amino acids, approximately 45 to approximately 80 amino acids, approximately 45 to approximately 70 amino acids, approximately 45 to approximately 60 amino acids, approximately 45 to approximately 50 amino acids, approximately 50 to approximately 100 amino acids, approximately 50 to approximately 90 amino acids, approximately 50 to approximately 80 amino acids, approximately 50 to approximately 70 amino acids, approximately 50 to approximately 60 amino acids, approximately 60 to approximately 100 amino acids.Approximately 60 to approximately 90 amino acids, approximately 60 to approximately 80 amino acids, approximately 60 to approximately 70 amino acids, approximately 70 to approximately 100 amino acids, approximately 70 to approximately 90 amino acids, approximately 70 to approximately 80 amino acids, approximately 80 to approximately 100 amino acids, approximately 80 to approximately 90 amino acids, or approximately 90 to approximately 100 amino acids.

[0144] In some embodiments, the linker sequence is rich in glycine (Gly or G) residues. In some embodiments, the linker sequence is rich in serine (Ser or S) residues. In some embodiments, the linker sequence is rich in both glycine and serine residues. In some embodiments, the linker sequence has one or more glycine-serine residue pairs (GS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker sequence has one or more Gly-Gly-Gly-Ser (GGGS) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker sequence has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the connector sequence has one or more Gly-Gly-Ser-Gly (GGSG) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

[0145] In some embodiments, the adapter sequence may include or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 11). In some embodiments, the adapter sequence may be encoded by nucleic acids including or consisting of: GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 12). In some embodiments, the adapter sequence may include or consist of: GGGSGGGS (SEQ ID NO: 13).

[0146] Target-binding domain

[0147] In some embodiments of any first multi-chain chimeric polypeptide or any second multi-chain chimeric polypeptide described herein, the first and second target-binding domains may be antigen-binding domains (e.g., any exemplary antigen-binding domain described herein or known in the art, such as an agonist antigen-binding domain), or soluble interleukin or cytokine proteins (e.g., any exemplary soluble interleukin protein described herein or known in the art).

[0148] In some embodiments of any of the first multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain each independently and specifically bind to the IL-18 receptor and the IL-12 receptor.

[0149] In some embodiments of any second multi-chain chimeric polypeptide described herein, the first target-binding domain and the second target-binding domain each independently and specifically bind to the IL-7 receptor and the IL-21 receptor.

[0150] In some embodiments of any first or second multi-chain chimeric polypeptide described herein, the first target-binding domain and / or the second target-binding domain may be a soluble interleukin or a soluble cytokine protein. In some instances, the soluble interleukin or soluble cytokine protein is selected from the group consisting of IL-7, IL-12, IL-18, and IL-21.

[0151] In some embodiments of any first multi-chain chimeric polypeptide or any second multi-chain chimeric polypeptide described herein, the first and second target-binding domains may each independently have about 5 amino acids to about 1000 amino acids, about 5 amino acids to about 950 amino acids, about 5 amino acids to about 900 amino acids, about 5 amino acids to about 850 amino acids, about 5 amino acids to about 800 amino acids, about 5 amino acids to about 750 amino acids, about 5 amino acids to about 700 amino acids, about 5 amino acids to about 650 amino acids, about 5 amino acids to about 600 amino acids, about 5 amino acids to about 550 amino acids, about 5 amino acids to about 500 amino acids, about 5 amino acids to about 450 amino acids, about 5 From approximately 400 amino acids, approximately 5 amino acids to approximately 350 amino acids, approximately 5 amino acids to approximately 300 amino acids, approximately 5 amino acids to approximately 280 amino acids, approximately 5 amino acids to approximately 260 amino acids, approximately 5 amino acids to approximately 240 amino acids, approximately 5 amino acids to approximately 220 amino acids, approximately 5 amino acids to approximately 200 amino acids, approximately 5 amino acids to approximately 195 amino acids, approximately 5 amino acids to approximately 190 amino acids, approximately 5 amino acids to approximately 185 amino acids, approximately 5 amino acids to approximately 180 amino acids, approximately 5 amino acids to approximately 175 amino acids, approximately 5 amino acids to approximately 170 amino acids, approximately 5 amino acids to approximately 165 amino acids, approximately 5 amino acids to approximately 160 amino acids. Amino acids, approximately 5 amino acids to approximately 155 amino acids, approximately 5 amino acids to approximately 150 amino acids, approximately 5 amino acids to approximately 145 amino acids, approximately 5 amino acids to approximately 140 amino acids, approximately 5 amino acids to approximately 135 amino acids, approximately 5 amino acids to approximately 130 amino acids, approximately 5 amino acids to approximately 125 amino acids, approximately 5 amino acids to approximately 120 amino acids, approximately 5 amino acids to approximately 115 amino acids, approximately 5 amino acids to approximately 110 amino acids, approximately 5 amino acids to approximately 105 amino acids, approximately 5 amino acids to approximately 100 amino acids, approximately 5 amino acids to approximately 95 amino acids, approximately 5 amino acids to approximately 90 amino acids, approximately 5 amino acids to approximately 85 amino acids, approximately 5 amino acids to approximately 8 0 amino acids, approximately 5 amino acids to approximately 75 amino acids, approximately 5 amino acids to approximately 70 amino acids, approximately 5 amino acids to approximately 65 amino acids, approximately 5 amino acids to approximately 60 amino acids, approximately 5 amino acids to approximately 55 amino acids, approximately 5 amino acids to approximately 50 amino acids, approximately 5 amino acids to approximately 45 amino acids, approximately 5 amino acids to approximately 40 amino acids, approximately 5 amino acids to approximately 35 amino acids, approximately 5 amino acids to approximately 30 amino acids, approximately 5 amino acids to approximately 25 amino acids, approximately 5 amino acids to approximately 20 amino acids, approximately 5 amino acids to approximately 15 amino acids, approximately 5 amino acids to approximately 10 amino acids, approximately 10 amino acids to approximately 1000 amino acids, approximately 10 amino acids to approximately 950 amino acids.From approximately 10 amino acids to approximately 900 amino acids, from approximately 10 amino acids to approximately 850 amino acids, from approximately 10 amino acids to approximately 800 amino acids, from approximately 10 amino acids to approximately 750 amino acids, from approximately 10 amino acids to approximately 700 amino acids, from approximately 10 amino acids to approximately 650 amino acids, from approximately 10 amino acids to approximately 600 amino acids, from approximately 10 amino acids to approximately 550 amino acids, from approximately 10 amino acids to approximately 500 amino acids, from approximately 10 amino acids to approximately 450 amino acids, from approximately 10 amino acids to approximately 400 amino acids, from approximately 10 amino acids to approximately 350 amino acids, from approximately 10 amino acids to approximately 300 amino acids, from approximately 10 amino acids to approximately 280 amino acids, from approximately 10 amino acids to... Approximately 260 amino acids, approximately 10 amino acids to approximately 240 amino acids, approximately 10 amino acids to approximately 220 amino acids, approximately 10 amino acids to approximately 200 amino acids, approximately 10 amino acids to approximately 195 amino acids, approximately 10 amino acids to approximately 190 amino acids, approximately 10 amino acids to approximately 185 amino acids, approximately 10 amino acids to approximately 180 amino acids, approximately 10 amino acids to approximately 175 amino acids, approximately 10 amino acids to approximately 170 amino acids, approximately 10 amino acids to approximately 165 amino acids, approximately 10 amino acids to approximately 160 amino acids, approximately 10 amino acids to approximately 155 amino acids, approximately 10 amino acids to approximately 150 amino acids, approximately 10 amino acids to approximately 145 amino acids. Approximately 10 amino acids to approximately 140 amino acids, approximately 10 amino acids to approximately 135 amino acids, approximately 10 amino acids to approximately 130 amino acids, approximately 10 amino acids to approximately 125 amino acids, approximately 10 amino acids to approximately 120 amino acids, approximately 10 amino acids to approximately 115 amino acids, approximately 10 amino acids to approximately 110 amino acids, approximately 10 amino acids to approximately 105 amino acids, approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 95 amino acids, approximately 10 amino acids to approximately 90 amino acids, approximately 10 amino acids to approximately 85 amino acids, approximately 10 amino acids to approximately 80 amino acids, approximately 10 amino acids to approximately 75 amino acids, approximately 10 amino acids to approximately 70 amino acids. Acids, approximately 10 to approximately 65 amino acids, approximately 10 to approximately 60 amino acids, approximately 10 to approximately 55 amino acids, approximately 10 to approximately 50 amino acids, approximately 10 to approximately 45 amino acids, approximately 10 to approximately 40 amino acids, approximately 10 to approximately 35 amino acids, approximately 10 to approximately 30 amino acids, approximately 10 to approximately 25 amino acids, approximately 10 to approximately 20 amino acids, approximately 10 to approximately 15 amino acids, approximately 15 to approximately 1000 amino acids, approximately 15 to approximately 950 amino acids, approximately 15 to approximately 900 amino acids, approximately 15 to approximately 850 amino acids.From approximately 15 amino acids to approximately 800 amino acids, from approximately 15 amino acids to approximately 750 amino acids, from approximately 15 amino acids to approximately 700 amino acids, from approximately 15 amino acids to approximately 650 amino acids, from approximately 15 amino acids to approximately 600 amino acids, from approximately 15 amino acids to approximately 550 amino acids, from approximately 15 amino acids to approximately 500 amino acids, from approximately 15 amino acids to approximately 450 amino acids, from approximately 15 amino acids to approximately 400 amino acids, from approximately 15 amino acids to approximately 350 amino acids, from approximately 15 amino acids to approximately 300 amino acids, from approximately 15 amino acids to approximately 280 amino acids, from approximately 15 amino acids to approximately 260 amino acids, from approximately 15 amino acids to approximately 240 amino acids, from approximately 15 amino acids to approximately... 220 amino acids, approximately 15 amino acids to approximately 200 amino acids, approximately 15 amino acids to approximately 195 amino acids, approximately 15 amino acids to approximately 190 amino acids, approximately 15 amino acids to approximately 185 amino acids, approximately 15 amino acids to approximately 180 amino acids, approximately 15 amino acids to approximately 175 amino acids, approximately 15 amino acids to approximately 170 amino acids, approximately 15 amino acids to approximately 165 amino acids, approximately 15 amino acids to approximately 160 amino acids, approximately 15 amino acids to approximately 155 amino acids, approximately 15 amino acids to approximately 150 amino acids, approximately 15 amino acids to approximately 145 amino acids, approximately 15 amino acids to approximately 140 amino acids, approximately 15 amino acids to approximately 135 amino acids, approximately From 15 amino acids to about 130 amino acids, from about 15 amino acids to about 125 amino acids, from about 15 amino acids to about 120 amino acids, from about 15 amino acids to about 115 amino acids, from about 15 amino acids to about 110 amino acids, from about 15 amino acids to about 105 amino acids, from about 15 amino acids to about 100 amino acids, from about 15 amino acids to about 95 amino acids, from about 15 amino acids to about 90 amino acids, from about 15 amino acids to about 85 amino acids, from about 15 amino acids to about 80 amino acids, from about 15 amino acids to about 75 amino acids, from about 15 amino acids to about 70 amino acids, from about 15 amino acids to about 65 amino acids, from about 15 amino acids to about 60 amino acids, and so on. From 15 amino acids to approximately 55 amino acids, from approximately 15 amino acids to approximately 50 amino acids, from approximately 15 amino acids to approximately 45 amino acids, from approximately 15 amino acids to approximately 40 amino acids, from approximately 15 amino acids to approximately 35 amino acids, from approximately 15 amino acids to approximately 30 amino acids, from approximately 15 amino acids to approximately 25 amino acids, from approximately 15 amino acids to approximately 20 amino acids, from approximately 20 amino acids to approximately 1000 amino acids, from approximately 20 amino acids to approximately 950 amino acids, from approximately 20 amino acids to approximately 900 amino acids, from approximately 20 amino acids to approximately 850 amino acids, from approximately 20 amino acids to approximately 800 amino acids, from approximately 20 amino acids to approximately 750 amino acids, from approximately 20 amino acids to approximately 700 amino acids.From approximately 20 amino acids to approximately 650 amino acids, from approximately 20 amino acids to approximately 600 amino acids, from approximately 20 amino acids to approximately 550 amino acids, from approximately 20 amino acids to approximately 500 amino acids, from approximately 20 amino acids to approximately 450 amino acids, from approximately 20 amino acids to approximately 400 amino acids, from approximately 20 amino acids to approximately 350 amino acids, from approximately 20 amino acids to approximately 300 amino acids, from approximately 20 amino acids to approximately 280 amino acids, from approximately 20 amino acids to approximately 260 amino acids, from approximately 20 amino acids to approximately 240 amino acids, from approximately 20 amino acids to approximately 220 amino acids, from approximately 20 amino acids to approximately 200 amino acids, from approximately 20 amino acids to approximately 195 amino acids, from approximately 20 amino acids to approximately... 190 amino acids, approximately 20 amino acids to approximately 185 amino acids, approximately 20 amino acids to approximately 180 amino acids, approximately 20 amino acids to approximately 175 amino acids, approximately 20 amino acids to approximately 170 amino acids, approximately 20 amino acids to approximately 165 amino acids, approximately 20 amino acids to approximately 160 amino acids, approximately 20 amino acids to approximately 155 amino acids, approximately 20 amino acids to approximately 150 amino acids, approximately 20 amino acids to approximately 145 amino acids, approximately 20 amino acids to approximately 140 amino acids, approximately 20 amino acids to approximately 135 amino acids, approximately 20 amino acids to approximately 130 amino acids, approximately 20 amino acids to approximately 125 amino acids, approximately 20 amino acids to approximately 120 amino acids, approximately From 20 amino acids to approximately 115 amino acids, from approximately 20 amino acids to approximately 110 amino acids, from approximately 20 amino acids to approximately 105 amino acids, from approximately 20 amino acids to approximately 100 amino acids, from approximately 20 amino acids to approximately 95 amino acids, from approximately 20 amino acids to approximately 90 amino acids, from approximately 20 amino acids to approximately 85 amino acids, from approximately 20 amino acids to approximately 80 amino acids, from approximately 20 amino acids to approximately 75 amino acids, from approximately 20 amino acids to approximately 70 amino acids, from approximately 20 amino acids to approximately 65 amino acids, from approximately 20 amino acids to approximately 60 amino acids, from approximately 20 amino acids to approximately 55 amino acids, from approximately 20 amino acids to approximately 50 amino acids, from approximately 20 amino acids to approximately 45 amino acids, from approximately 20 amino acids... From approximately 40 amino acids, from approximately 20 amino acids to approximately 35 amino acids, from approximately 20 amino acids to approximately 30 amino acids, from approximately 20 amino acids to approximately 25 amino acids, from approximately 25 amino acids to approximately 1000 amino acids, from approximately 25 amino acids to approximately 950 amino acids, from approximately 25 amino acids to approximately 900 amino acids, from approximately 25 amino acids to approximately 850 amino acids, from approximately 25 amino acids to approximately 800 amino acids, from approximately 25 amino acids to approximately 750 amino acids, from approximately 25 amino acids to approximately 700 amino acids, from approximately 25 amino acids to approximately 650 amino acids, from approximately 25 amino acids to approximately 600 amino acids, from approximately 25 amino acids to approximately 550 amino acids, from approximately 25 amino acids to approximately 500 amino acids.From approximately 25 amino acids to approximately 450 amino acids, from approximately 25 amino acids to approximately 400 amino acids, from approximately 25 amino acids to approximately 350 amino acids, from approximately 25 amino acids to approximately 300 amino acids, from approximately 25 amino acids to approximately 280 amino acids, from approximately 25 amino acids to approximately 260 amino acids, from approximately 25 amino acids to approximately 240 amino acids, from approximately 25 amino acids to approximately 220 amino acids, from approximately 25 amino acids to approximately 200 amino acids, from approximately 25 amino acids to approximately 195 amino acids, from approximately 25 amino acids to approximately 190 amino acids, from approximately 25 amino acids to approximately 185 amino acids, from approximately 25 amino acids to approximately 180 amino acids, from approximately 25 amino acids to approximately 175 amino acids, from approximately 25 amino acids to approximately... 170 amino acids, approximately 25 amino acids to approximately 165 amino acids, approximately 25 amino acids to approximately 160 amino acids, approximately 25 amino acids to approximately 155 amino acids, approximately 25 amino acids to approximately 150 amino acids, approximately 25 amino acids to approximately 145 amino acids, approximately 25 amino acids to approximately 140 amino acids, approximately 25 amino acids to approximately 135 amino acids, approximately 25 amino acids to approximately 130 amino acids, approximately 25 amino acids to approximately 125 amino acids, approximately 25 amino acids to approximately 120 amino acids, approximately 25 amino acids to approximately 115 amino acids, approximately 25 amino acids to approximately 110 amino acids, approximately 25 amino acids to approximately 105 amino acids, approximately 25 amino acids to approximately 100 amino acids, approximately 2 From 5 amino acids to approximately 95 amino acids, from approximately 25 amino acids to approximately 90 amino acids, from approximately 25 amino acids to approximately 85 amino acids, from approximately 25 amino acids to approximately 80 amino acids, from approximately 25 amino acids to approximately 75 amino acids, from approximately 25 amino acids to approximately 70 amino acids, from approximately 25 amino acids to approximately 65 amino acids, from approximately 25 amino acids to approximately 60 amino acids, from approximately 25 amino acids to approximately 55 amino acids, from approximately 25 amino acids to approximately 50 amino acids, from approximately 25 amino acids to approximately 45 amino acids, from approximately 25 amino acids to approximately 40 amino acids, from approximately 25 amino acids to approximately 35 amino acids, from approximately 25 amino acids to approximately 30 amino acids, from approximately 30 amino acids to approximately 1000 amino acids, from approximately 30 amino acids. From approximately 950 amino acids, from approximately 30 amino acids to approximately 900 amino acids, from approximately 30 amino acids to approximately 850 amino acids, from approximately 30 amino acids to approximately 800 amino acids, from approximately 30 amino acids to approximately 750 amino acids, from approximately 30 amino acids to approximately 700 amino acids, from approximately 30 amino acids to approximately 650 amino acids, from approximately 30 amino acids to approximately 600 amino acids, from approximately 30 amino acids to approximately 550 amino acids, from approximately 30 amino acids to approximately 500 amino acids, from approximately 30 amino acids to approximately 450 amino acids, from approximately 30 amino acids to approximately 400 amino acids, from approximately 30 amino acids to approximately 350 amino acids, from approximately 30 amino acids to approximately 300 amino acids, from approximately 30 amino acids to approximately 280 amino acids.From approximately 30 amino acids to approximately 260 amino acids, from approximately 30 amino acids to approximately 240 amino acids, from approximately 30 amino acids to approximately 220 amino acids, from approximately 30 amino acids to approximately 200 amino acids, from approximately 30 amino acids to approximately 195 amino acids, from approximately 30 amino acids to approximately 190 amino acids, from approximately 30 amino acids to approximately 185 amino acids, from approximately 30 amino acids to approximately 180 amino acids, from approximately 30 amino acids to approximately 175 amino acids, from approximately 30 amino acids to approximately 170 amino acids, from approximately 30 amino acids to approximately 165 amino acids, from approximately 30 amino acids to approximately 160 amino acids, from approximately 30 amino acids to approximately 155 amino acids, from approximately 30 amino acids to approximately 150 amino acids, from approximately 30 amino acids to approximately... 145 amino acids, approximately 30 amino acids to approximately 140 amino acids, approximately 30 amino acids to approximately 135 amino acids, approximately 30 amino acids to approximately 130 amino acids, approximately 30 amino acids to approximately 125 amino acids, approximately 30 amino acids to approximately 120 amino acids, approximately 30 amino acids to approximately 115 amino acids, approximately 30 amino acids to approximately 110 amino acids, approximately 30 amino acids to approximately 105 amino acids, approximately 30 amino acids to approximately 100 amino acids, approximately 30 amino acids to approximately 95 amino acids, approximately 30 amino acids to approximately 90 amino acids, approximately 30 amino acids to approximately 85 amino acids, approximately 30 amino acids to approximately 80 amino acids, approximately 30 amino acids to approximately 75 amino acids, approximately 30 amino acids From approximately 70 amino acids, from approximately 30 amino acids to approximately 65 amino acids, from approximately 30 amino acids to approximately 60 amino acids, from approximately 30 amino acids to approximately 55 amino acids, from approximately 30 amino acids to approximately 50 amino acids, from approximately 30 amino acids to approximately 45 amino acids, from approximately 30 amino acids to approximately 40 amino acids, from approximately 30 amino acids to approximately 35 amino acids, from approximately 35 amino acids to approximately 1000 amino acids, from approximately 35 amino acids to approximately 950 amino acids, from approximately 35 amino acids to approximately 900 amino acids, from approximately 35 amino acids to approximately 850 amino acids, from approximately 35 amino acids to approximately 800 amino acids, from approximately 35 amino acids to approximately 750 amino acids, from approximately 35 amino acids to approximately 700 amino acids, from approximately 35 amino acids From approximately 650 amino acids, from approximately 35 amino acids to approximately 600 amino acids, from approximately 35 amino acids to approximately 550 amino acids, from approximately 35 amino acids to approximately 500 amino acids, from approximately 35 amino acids to approximately 450 amino acids, from approximately 35 amino acids to approximately 400 amino acids, from approximately 35 amino acids to approximately 350 amino acids, from approximately 35 amino acids to approximately 300 amino acids, from approximately 35 amino acids to approximately 280 amino acids, from approximately 35 amino acids to approximately 260 amino acids, from approximately 35 amino acids to approximately 240 amino acids, from approximately 35 amino acids to approximately 220 amino acids, from approximately 35 amino acids to approximately 200 amino acids, from approximately 35 amino acids to approximately 195 amino acids, from approximately 35 amino acids to approximately 190 amino acids.From approximately 35 amino acids to approximately 185 amino acids, from approximately 35 amino acids to approximately 180 amino acids, from approximately 35 amino acids to approximately 175 amino acids, from approximately 35 amino acids to approximately 170 amino acids, from approximately 35 amino acids to approximately 165 amino acids, from approximately 35 amino acids to approximately 160 amino acids, from approximately 35 amino acids to approximately 155 amino acids, from approximately 35 amino acids to approximately 150 amino acids, from approximately 35 amino acids to approximately 145 amino acids, from approximately 35 amino acids to approximately 140 amino acids, from approximately 35 amino acids to approximately 135 amino acids, from approximately 35 amino acids to approximately 130 amino acids, from approximately 35 amino acids to approximately 125 amino acids, from approximately 35 amino acids to approximately 120 amino acids, from approximately 35 amino acids to approximately 1... 15 amino acids, approximately 35 amino acids to approximately 110 amino acids, approximately 35 amino acids to approximately 105 amino acids, approximately 35 amino acids to approximately 100 amino acids, approximately 35 amino acids to approximately 95 amino acids, approximately 35 amino acids to approximately 90 amino acids, approximately 35 amino acids to approximately 85 amino acids, approximately 35 amino acids to approximately 80 amino acids, approximately 35 amino acids to approximately 75 amino acids, approximately 35 amino acids to approximately 70 amino acids, approximately 35 amino acids to approximately 65 amino acids, approximately 35 amino acids to approximately 60 amino acids, approximately 35 amino acids to approximately 55 amino acids, approximately 35 amino acids to approximately 50 amino acids, approximately 35 amino acids to approximately 45 amino acids, approximately 35 amino acids to approximately 40 amino acids. Acids, approximately 40 amino acids to approximately 1000 amino acids, approximately 40 amino acids to approximately 950 amino acids, approximately 40 amino acids to approximately 900 amino acids, approximately 40 amino acids to approximately 850 amino acids, approximately 40 amino acids to approximately 800 amino acids, approximately 40 amino acids to approximately 750 amino acids, approximately 40 amino acids to approximately 700 amino acids, approximately 40 amino acids to approximately 650 amino acids, approximately 40 amino acids to approximately 600 amino acids, approximately 40 amino acids to approximately 550 amino acids, approximately 40 amino acids to approximately 500 amino acids, approximately 40 amino acids to approximately 450 amino acids, approximately 40 amino acids to approximately 400 amino acids, approximately 40 amino acids to approximately 350 amino acids, approximately 40 amino acids From approximately 300 amino acids, from approximately 40 amino acids to approximately 280 amino acids, from approximately 40 amino acids to approximately 260 amino acids, from approximately 40 amino acids to approximately 240 amino acids, from approximately 40 amino acids to approximately 220 amino acids, from approximately 40 amino acids to approximately 200 amino acids, from approximately 40 amino acids to approximately 195 amino acids, from approximately 40 amino acids to approximately 190 amino acids, from approximately 40 amino acids to approximately 185 amino acids, from approximately 40 amino acids to approximately 180 amino acids, from approximately 40 amino acids to approximately 175 amino acids, from approximately 40 amino acids to approximately 170 amino acids, from approximately 40 amino acids to approximately 165 amino acids, from approximately 40 amino acids to approximately 160 amino acids, from approximately 40 amino acids to approximately 155 amino acids.Approximately 40 to approximately 150 amino acids, approximately 40 to approximately 145 amino acids, approximately 40 to approximately 140 amino acids, approximately 40 to approximately 135 amino acids, approximately 40 to approximately 130 amino acids, approximately 40 to approximately 125 amino acids, approximately 40 to approximately 120 amino acids, approximately 40 to approximately 115 amino acids, approximately 40 to approximately 110 amino acids, approximately 40 to approximately 105 amino acids, approximately 40 to approximately 100 amino acids, approximately 40 to approximately 95 amino acids, approximately 40 to approximately 90 amino acids, approximately 40 to approximately 85 amino acids, approximately 40 to approximately 80 amino acids. Amino acids, approximately 40 to approximately 75 amino acids, approximately 40 to approximately 70 amino acids, approximately 40 to approximately 65 amino acids, approximately 40 to approximately 60 amino acids, approximately 40 to approximately 55 amino acids, approximately 40 to approximately 50 amino acids, approximately 40 to approximately 45 amino acids, approximately 45 to approximately 1000 amino acids, approximately 45 to approximately 950 amino acids, approximately 45 to approximately 900 amino acids, approximately 45 to approximately 850 amino acids, approximately 45 to approximately 800 amino acids, approximately 45 to approximately 750 amino acids, approximately 45 to approximately 700 amino acids, approximately 45 to approximately 650 amino acids Amino acids, approximately 45 amino acids to approximately 600 amino acids, approximately 45 amino acids to approximately 550 amino acids, approximately 45 amino acids to approximately 500 amino acids, approximately 45 amino acids to approximately 450 amino acids, approximately 45 amino acids to approximately 400 amino acids, approximately 45 amino acids to approximately 350 amino acids, approximately 45 amino acids to approximately 300 amino acids, approximately 45 amino acids to approximately 280 amino acids, approximately 45 amino acids to approximately 260 amino acids, approximately 45 amino acids to approximately 240 amino acids, approximately 45 amino acids to approximately 220 amino acids, approximately 45 amino acids to approximately 200 amino acids, approximately 45 amino acids to approximately 195 amino acids, approximately 45 amino acids to approximately 190 amino acids, approximately 45 amino groups. From approximately 185 amino acids, from approximately 45 amino acids to approximately 180 amino acids, from approximately 45 amino acids to approximately 175 amino acids, from approximately 45 amino acids to approximately 170 amino acids, from approximately 45 amino acids to approximately 165 amino acids, from approximately 45 amino acids to approximately 160 amino acids, from approximately 45 amino acids to approximately 155 amino acids, from approximately 45 amino acids to approximately 150 amino acids, from approximately 45 amino acids to approximately 145 amino acids, from approximately 45 amino acids to approximately 140 amino acids, from approximately 45 amino acids to approximately 135 amino acids, from approximately 45 amino acids to approximately 130 amino acids, from approximately 45 amino acids to approximately 125 amino acids, from approximately 45 amino acids to approximately 120 amino acids, from approximately 45 amino acids to approximately 115 amino acids.Approximately 45 amino acids to approximately 110 amino acids, approximately 45 amino acids to approximately 105 amino acids, approximately 45 amino acids to approximately 100 amino acids, approximately 45 amino acids to approximately 95 amino acids, approximately 45 amino acids to approximately 90 amino acids, approximately 45 amino acids to approximately 85 amino acids, approximately 45 amino acids to approximately 80 amino acids, approximately 45 amino acids to approximately 75 amino acids, approximately 45 amino acids to approximately 70 amino acids, approximately 45 amino acids to approximately 65 amino acids, approximately 45 amino acids to approximately 60 amino acids, approximately 45 amino acids to approximately 55 amino acids, approximately 45 amino acids to approximately 50 amino acids, approximately 50 amino acids to approximately 1000 amino acids, approximately 50 amino acids to approximately 950 amino acids, approximately From 50 amino acids to approximately 900 amino acids, from approximately 50 amino acids to approximately 850 amino acids, from approximately 50 amino acids to approximately 800 amino acids, from approximately 50 amino acids to approximately 750 amino acids, from approximately 50 amino acids to approximately 700 amino acids, from approximately 50 amino acids to approximately 650 amino acids, from approximately 50 amino acids to approximately 600 amino acids, from approximately 50 amino acids to approximately 550 amino acids, from approximately 50 amino acids to approximately 500 amino acids, from approximately 50 amino acids to approximately 450 amino acids, from approximately 50 amino acids to approximately 400 amino acids, from approximately 50 amino acids to approximately 350 amino acids, from approximately 50 amino acids to approximately 300 amino acids, from approximately 50 amino acids to approximately 280 amino acids, from approximately 50 amino acids to approximately 2... 60 amino acids, approximately 50 amino acids to approximately 240 amino acids, approximately 50 amino acids to approximately 220 amino acids, approximately 50 amino acids to approximately 200 amino acids, approximately 50 amino acids to approximately 195 amino acids, approximately 50 amino acids to approximately 190 amino acids, approximately 50 amino acids to approximately 185 amino acids, approximately 50 amino acids to approximately 180 amino acids, approximately 50 amino acids to approximately 175 amino acids, approximately 50 amino acids to approximately 170 amino acids, approximately 50 amino acids to approximately 165 amino acids, approximately 50 amino acids to approximately 160 amino acids, approximately 50 amino acids to approximately 155 amino acids, approximately 50 amino acids to approximately 150 amino acids, approximately 50 amino acids to approximately 145 amino acids, approximately 5 From 0 amino acids to approximately 140 amino acids, from approximately 50 amino acids to approximately 135 amino acids, from approximately 50 amino acids to approximately 130 amino acids, from approximately 50 amino acids to approximately 125 amino acids, from approximately 50 amino acids to approximately 120 amino ...

Claims

1. An in vitro method for promoting the activation and proliferation of natural killer cells (NK cells), the method comprising: (a) The natural killer cells are contacted in a liquid culture medium containing an effective amount of the first multi-chain chimeric polypeptide for 15 minutes to 1 day under conditions that allow for differentiation of natural killer cells; as well as (b) The natural killer cells are contacted in a liquid culture medium comprising effective amounts of (i) a second multi-chain chimeric polypeptide and (ii) an IgG1 antibody construct for 1 to 30 days, under conditions that allow for activation and proliferation of the natural killer cells. The first multi-chain chimeric polypeptide is composed of a first chimeric polypeptide and a second chimeric polypeptide. The amino acid sequence of the first chimeric polypeptide in the first multi-chain chimeric polypeptide is SEQ ID NO:63; and The amino acid sequence of the second chimeric polypeptide in the first multi-chain chimeric polypeptide is SEQ ID NO:65; and The second multi-chain chimeric polypeptide is composed of the first chimeric polypeptide and the second chimeric polypeptide. The amino acid sequence of the first chimeric polypeptide in the second multi-chain chimeric polypeptide is SEQ ID NO:67, and the amino acid sequence of the second chimeric polypeptide in the second multi-chain chimeric polypeptide is SEQ ID NO:

69. The IgG1 antibody construct includes at least one antigen-binding domain that specifically binds to a soluble tissue factor domain.

2. The method of claim 1, wherein the first multi-chain chimeric polypeptide does not stimulate blood clotting in mammals.

3. The method according to claim 1 or 2, wherein the second multi-chain chimeric polypeptide does not stimulate blood clotting in mammals.

4. The method according to claim 1 or 2, wherein the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the soluble tissue factor domain.

5. The method according to claim 1 or 2, wherein the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the soluble tissue factor domain.

6. The method according to claim 1 or 2, wherein the contact in step (a) is performed for 15 minutes to 4 hours.

7. The method of claim 6, wherein the contact in step (a) is performed for 30 minutes to 3.5 hours.

8. The method of claim 7, wherein the contact in step (a) is performed for 1 to 3.5 hours.

9. The method of claim 8, wherein the contact in step (a) is performed for 2 to 3.5 hours.

10. The method according to any one of claims 1-2 and 7-9, wherein the liquid culture medium in step (a) and / or step (b) is a serum-containing liquid culture medium.

11. The method according to any one of claims 1-2 and 7-9, wherein the NK cells are at a density of 4.0 x 10⁻⁶. 7 1.5 x 10 cells / mL to 1.5 x 10 8 A density of cells / mL is present in the liquid culture medium in step (a).

12. The method according to any one of claims 1-2 and 7-9, wherein the NK cells are at a concentration of 5.0 x 10⁻⁶. 7 cells / mL to 2.0 x 10⁻⁶ 8 A density of cells / mL is present in the liquid culture medium in step (a).

13. The method according to any one of claims 1-2 and 7-9, wherein the contact in step (b) is carried out for 7 to 30 days.

14. The method of claim 13, wherein the contact in step (b) is performed for 14 to 21 days.

15. The method according to any one of claims 1-2, 7-9 and 14, wherein the liquid culture medium in step (b) comprises the second multi-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of 0.5:1 to 2:

1.

16. The method of claim 15, wherein the liquid culture medium in step (b) comprises the second multi-chain chimeric polypeptide and the IgG1 antibody construct in a molar ratio of 0.8:1 to 1.2:

1.

17. The method according to any one of claims 1-2, 7-9, 14, and 16, wherein the NK cells are maintained at 0.5 x 10⁻⁶ in the liquid culture medium in step (b). 6 cells / mL to 2.0 x 10⁻⁶ 6 Density of cells per milliliter.

18. The method of claim 17, wherein the NK cells are maintained at 1.0 x 10⁻⁶ in the liquid culture medium in step (b). 6 cells / mL to 2.0 x 10⁻⁶ 6 Density of cells per milliliter.

19. The method according to any one of claims 1-2, 7-9, 14, 16 and 18, wherein the NK cells were previously obtained from the subject.

20. The method according to any one of claims 1-2, 7-9, 14, 16 and 18, wherein the NK cells are previously obtained from umbilical cord blood.

21. The method according to any one of claims 1-2, 7-9, 14, 16 and 18, wherein the NK cells have been previously genetically modified to express chimeric antigen receptors or recombinant T cell receptors.

22. The method according to any one of claims 1-2, 7-9, 14, 16 and 18, wherein the method further comprises, between step (a) and step (b), introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells.

23. The method according to any one of claims 1-2, 7-9, 14, 16 and 18, wherein the method further comprises, prior to step (a), introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells.

24. The method according to any one of claims 1-2, 7-9, 14, 16 and 18, wherein the method further comprises, during step (b), introducing a nucleic acid encoding a chimeric antigen receptor or a recombinant T cell receptor into the NK cells.

25. The method according to any one of claims 1-2, 7-9, 14, 16 and 18, wherein the method further comprises isolating the NK cells after step (b).

26. A reagent kit comprising: (a) A first multi-chain chimeric polypeptide, wherein the first multi-chain chimeric polypeptide is composed of a first chimeric polypeptide and a second chimeric polypeptide. The amino acid sequence of the first chimeric polypeptide in the first multi-chain chimeric polypeptide is SEQ ID NO:63, and the amino acid sequence of the second chimeric polypeptide in the first multi-chain chimeric polypeptide is SEQ ID NO:

65. (b) A second multi-chain chimeric polypeptide, which is composed of a first chimeric polypeptide and a second chimeric polypeptide. The amino acid sequence of the first chimeric polypeptide in the second multi-chain chimeric polypeptide is SEQ ID NO:67, and the amino acid sequence of the second chimeric polypeptide in the second multi-chain chimeric polypeptide is SEQ ID NO:69; and (c) An IgG1 antibody construct comprising at least one antigen-binding domain that specifically binds to a soluble tissue factor domain.

27. The kit of claim 26, wherein the first multi-chain chimeric polypeptide does not stimulate blood clotting in mammals.

28. The kit according to claim 26 or 27, wherein the second multi-chain chimeric polypeptide does not stimulate blood clotting in mammals.

29. The kit according to claim 26 or 27, wherein the IgG1 antibody construct is a monoclonal IgG1 antibody, wherein both antigen-binding domains of the monoclonal IgG1 antibody specifically bind to the soluble tissue factor domain.

30. The kit according to claim 26 or 27, wherein the IgG1 antibody construct is a bispecific IgG1 antibody, wherein one of the two antigen-binding domains of the bispecific IgG1 antibody specifically binds to the soluble tissue factor domain.

Images