JPWO2020123938A5 - - Google Patents

Description

Generally, in the following claims, the language used should not be construed as limiting the claims to the particular embodiments disclosed in this specification, rather the claims to which such claims are entitled. It should be construed to include all possible embodiments along with the full range of equivalents to which it is entitled. Accordingly, the claims are not limited by the disclosure.
The present invention provides, for example, the following items.
(Item 1)
a non-natural cell,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a first co-stimulatory domain, and/or a primary signaling domain; and
(b) a second polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second co-stimulatory domain;
A bridging factor promotes formation of a polypeptide complex on said non-native cell surface, said bridging factor being associated with and disposed between said first and second multimerization domains. natural cells.
(Item 2)
2. The non-natural cell of item 1, wherein said first and second multimerization domains are different.
(Item 3)
3. The non-natural cell of item 1 or item 2, wherein said first and second co-stimulatory domains are different.
(Item 4)
Said first multimerization domain and said second multimerization domain are rapamycin or its rapalogs, coumarmycin or its derivatives, gibberellin or its derivatives, abscisic acid (ABA) or its derivatives, methotrexate or its derivatives, cyclosporine A or a derivative thereof, FK506/cyclosporin A (FKCsA) or a derivative thereof, and a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof. , items 1-3.
(Item 5)
The first multimerization domain and the second multimerization domain are FKBP and FKBP-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin , and a pair selected from the group consisting of PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
(Item 6)
6. The method of any one of items 1-5, wherein the first multimerization domain comprises a FKBP polypeptide or variant thereof and the second multimerization domain comprises a FRB polypeptide or variant thereof. non-natural cells.
(Item 7)
6. The method of any one of items 1-5, wherein the first multimerization domain comprises a FRB polypeptide or variant thereof and the second multimerization domain comprises a FKBP polypeptide or variant thereof. non-natural cells.
(Item 8)
8. The non-natural cell according to any one of items 1 to 7, wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. .
(Item 9)
9. The non-natural cell of any one of items 1-8, wherein said first and second multimerization domains are selected from FRB T2098L and FKBP12 and said cross-linking agent is sirolimus or AP21967.
(Item 10)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1) Non-natural cell according to any one of items 1-9, selected from a polypeptide selected from the group.
(Item 11)
Any of items 1 to 10, wherein the first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain. The non-natural cell according to item 1.
(Item 12)
12. The non-natural cell of any one of items 1-11, wherein said first transmembrane domain and said second transmembrane domain are different.
(Item 13)
13. The non-natural cell of any one of items 1-12, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 14)
Said first and second co-stimulatory domains are independently Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 ( CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Ｌｉｎｋｅｒ ｆｏｒ ａｃｔｉｖａｔｉｏｎ ｏｆ Ｔ－ｃｅｌｌｓ ｆａｍｉｌｙ ｍｅｍｂｅｒ １（ＬＡＴ）、ＳＨ２ Ｄｏｍａｉｎ－Ｃｏｎｔａｉｎｉｎｇ Ｌｅｕｋｏｃｙｔｅ Ｐｒｏｔｅｉｎ Ｏｆ ７６ ｋＤ（ＳＬＰ７６）、Ｔ ｃｅｌｌ ｒｅｃｅｐｔｏｒ ａｓｓｏｃｉａｔｅｄ ｔｒａｎｓｍｅｍｂｒａｎｅ ａｄａｐｔｏｒ １（ＴＲＡＴ１）、ＴＮＦＲ２、ＴＮＦＲＳ１４、ＴＮＦＲＳ１８、ＴＮＦＲＳ２５、およびｚｅｔａ ｃｈａｉｎ ｏｆ T cells
14. The non-natural cell according to any one of items 1 to 13, selected from co-stimulatory molecules selected from the group consisting of receptor associated protein kinase 70 (ZAP70).
(Item 15)
said first costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of CD28, CD134 and CD137, and said second costimulatory domain comprises CD28, CD278, TNFRS14, TNFRS18, TNFRS25 , OX40 or TNFR2.
(Item 16)
16. The non-natural cell of any one of items 1-15, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from OX40 or TNFR2.
(Item 17)
17. The non-natural cell of any one of items 1-16, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from OX40.
(Item 18)
17. The non-natural cell of any one of items 1-16, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from TNFR2.
(Item 19)
19. Any one of items 1-18, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. The non-natural cell according to .
(Item 20)
20. The non-natural cell of any one of items 1-19, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 21)
21. The non-natural cell of any one of items 1-20, wherein said extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.
(Item 22)
The extracellular binding domain may be camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single domain antibodies (sdAbs, camelid VHHs, nanobodies) or antigen-binding fragments thereof. , items 1-21.
(Item 23)
23. The non-natural cell of any one of items 1-22, wherein said extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.
(Item 24)
23. The non-natural cell of any one of items 1-22, wherein said extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.
(Item 25)
25. The non-natural cell of any one of items 1-24, wherein said extracellular binding domain comprises a scFv.
(Item 26)
24. The non-natural cell of any one of items 1-23, wherein said extracellular binding domain comprises one or more camelid VHH antibodies.
(Item 27)
wherein said extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand, item 27. The non-natural cell according to any one of 1-26.
(Item 28)
The extracellular binding domain is alpha folate receptor (FRα), α _V β ₆ Integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase type IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6 , CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), claudin 6 (CLDN6), claudin 18 isoform 2 (CLDN18.2), C-type lectin -like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T cell lymphoma-associated antigen 1 (CTAGE1), delta-like Lactanic 3, delta-like canonic 3 Epidermal growth factor receptor (EGFR), epithelial growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activation protein (FAP), Fc Receptor Like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), ganglioside G3 (G2), ganglioside G2 (G2) -3 (GPC3), EGFR family including ErbB2 (HER2), HER2 p95, IL-10Rα, IL-13Rα2, Kappa, cancer/testis antigen 2 (LAGE-1A), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, melanoma antigen recognized by T cel ls 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain related proteins A (MICA), MHC class I chain related proteins B (MICB), neural cell adhesion molecule (NCAM), cancer/testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor －ｌｉｋｅ ｏｒｐｈａｎ ｒｅｃｅｐｔｏｒ １（ＲＯＲ１）、ｓｙｎｏｖｉａｌ ｓａｒｃｏｍａ、Ｘ ｂｒｅａｋｐｏｉｎｔ ２（ＳＳＸ２）、サバイビン、ｔｕｍｏｒ ａｓｓｏｃｉａｔｅｄ ｇｌｙｃｏｐｒｏｔｅｉｎ ７２（ＴＡＧ７２）、ｔｕｍｏｒ ｅｎｄｏｔｈｅｌｉａｌ ｍａｒｋｅｒ １（ＴＥＭ１／ＣＤ２４８）、ｔｕｍｏｒ ｅｎｄｏｔｈｅｌｉａｌ ｍａｒｋｅｒ ７－ｒｅｌａｔｅｄ（ＴＥＭ７Ｒ）、ｔｒｏｐｈｏｂｌａｓｔ selected from the group consisting of glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial cell growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1) 28. The non-natural cell according to any one of items 1-27, wherein the non-natural cell binds to an antigen to be administered.
(Item 29)
a non-natural cell,
(a) a first polypeptide comprising an FK506 binding protein (FKBP) multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and
(b) an antibody or antigen-binding fragment thereof, a FKBP-rapamycin binding (FRB) multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain; ,
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 30)
a non-natural cell,
(a) a first polypeptide comprising a FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and
(b) an antibody or antigen-binding fragment thereof, a FRB multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising a TNFR2 co-stimulatory domain;
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 31)
a non-natural cell,
(a) a first polypeptide comprising a FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and
(b) an antibody or antigen-binding fragment thereof, a FKBP multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain;
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 32)
a non-natural cell,
(a) a first polypeptide comprising a FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain; and
(b) an antibody or antigen-binding fragment thereof, a FKBP multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising a TNFR2 co-stimulatory domain;
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 33)
33. The non-natural cell of any one of items 29-32, wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. .
(Item 34)
34. The non-natural cell according to any one of items 29 to 33, wherein the FRB multimerization domain is FRB T2098L, the FKBP multimerization domain is FKBP12, and the cross-linking agent is sirolimus or AP21967. .
(Item 35)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1. 35. The non-natural cell according to any one of 29-34.
(Item 36)
Any of items 29 to 35, wherein said first transmembrane domain and said second transmembrane domain are independently selected from polypeptides selected from the group consisting of a CD4 transmembrane domain and a CD8α transmembrane domain. The non-natural cell according to item 1.
(Item 37)
37. Non-natural cell according to any one of items 29-36, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 38)
said co-stimulatory domain is Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27; CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Linker for activation of T-cells family member １（ＬＡＴ）、ＳＨ２ Ｄｏｍａｉｎ－Ｃｏｎｔａｉｎｉｎｇ Ｌｅｕｋｏｃｙｔｅ Ｐｒｏｔｅｉｎ Ｏｆ ７６ ｋＤ（ＳＬＰ７６）、Ｔ ｃｅｌｌ ｒｅｃｅｐｔｏｒ ａｓｓｏｃｉａｔｅｄ ｔｒａｎｓｍｅｍｂｒａｎｅ ａｄａｐｔｏｒ １（ＴＲＡＴ１）、ＴＮＦＲ２、ＴＮＦＲＳ１４、ＴＮＦＲＳ１８、ＴＮＦＲＳ２５、およびｚｅｔａ ｃｈａｉｎ ｏｆ Ｔ ｃｅｌｌ ｒｅｃｅｐｔｏｒ ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ ｋｉｎａｓｅ ７０（ＺＡＰ７０ 38. The non-natural cell according to any one of items 29-37, wherein the non-natural cell is isolated from a co-stimulatory molecule selected from the group consisting of:
(Item 39)
39. The non-natural cell of any one of items 29-38, wherein said co-stimulatory domain is isolated from a co-stimulatory molecule selected from the group consisting of CD28, CD134 and CD137.
(Item 40)
40. The non-natural cell of any one of items 29-39, wherein said costimulatory domain is isolated from a CD137 costimulatory molecule.
(Item 41)
41. Any one of items 29-40, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d. Non-natural cells as described in.
(Item 42)
42. The non-natural cell of any one of items 29-41, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 43)
The antibody or antigen-binding fragment thereof may be camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv), and single domain antibodies (sdAb, camelid VHH, nanobodies). The non-natural cell according to item 1.
(Item 44)
44. The non-natural cell of any one of items 29-43, wherein said antibody or antigen-binding fragment thereof is human or humanized.
(Item 45)
45. The non-natural cell of any one of items 29-44, wherein said antibody or antigen-binding fragment thereof comprises a scFv or one or more camelid VHH antibodies.
(Item 46)
The antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand. , items 29-45.
(Item 47)
The antibody or antigen-binding fragment thereof is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 47. Non-natural cell according to any one of items 29-46, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 48)
48. The non-natural cell of any one of items 29-47, wherein said antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B and/or EGFRvIII.
(Item 49)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to BCMA.
(Item 50)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to CD19.
(Item 51)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to CD20 or CD22.
(Item 52)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to B7-H3.
(Item 53)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to CD33.
(Item 54)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to CD79A.
(Item 55)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to CD79B.
(Item 56)
49. The non-natural cell of any one of items 29-48, wherein said antibody or antigen-binding fragment thereof binds to EGFRvIII.
(Item 57)
a non-natural cell,
(a) a first polypeptide comprising a FKBP12 multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain; and
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB T2098L multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a TNFR2 co-stimulatory domain;
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 58)
a non-natural cell,
(a) a first polypeptide comprising a FKBP12 multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain; and
(b) an antibody or antigen-binding fragment thereof, a FRB T2098L multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain;
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 59)
a non-natural cell,
(a) a first polypeptide comprising a FRB T2098L multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain, and
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP12 multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a TNFR2 co-stimulatory domain;
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 60)
a non-natural cell,
(a) a first polypeptide comprising a FRB T2098L multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain, and
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP12 multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and an OX40 co-stimulatory domain;
A bridging agent promotes formation of a polypeptide complex on said non-native cell surface, said bridging agent being associated with and disposed between said first and second polypeptide multimerization domains. , non-natural cells.
(Item 61)
61. The non-natural cell of any one of items 57-60, wherein said cross-linking agent is AP21967 or sirolimus.
(Item 62)
The antibody or antigen-binding fragment thereof may be camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv), and single domain antibodies (sdAb, camelid VHH, nanobodies). The non-natural cell according to item 1.
(Item 63)
63. The non-natural cell of any one of items 57-62, wherein said antibody or antigen-binding fragment thereof is human or humanized.
(Item 64)
64. The non-natural cell of any one of items 57-63, wherein said antibody or antigen-binding fragment thereof comprises a scFv or one or more camelid VHH antibodies.
(Item 65)
The antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand. , items 57-64.
(Item 66)
The antibody or antigen-binding fragment thereof is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 66. The non-natural cell of any one of items 57-65, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 67)
67. The non-natural cell of any one of items 57-66, wherein said antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B and/or EGFRvIII.
(Item 68)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to BCMA.
(Item 69)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to CD19.
(Item 70)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to CD20 or CD22.
(Item 71)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to B7H3.
(Item 72)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to CD33.
(Item 73)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to CD79A.
(Item 74)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to CD79B.
(Item 75)
68. The non-natural cell of any one of items 57-67, wherein said antibody or antigen-binding fragment thereof binds to EGFRvIII, optionally said antibody is EGFR806 or an antigen-binding fragment thereof.
(Item 76)
76. The non-natural cell according to any one of items 1 to 75, wherein said multimerization domain is extracellularly localized when said first polypeptide and said second polypeptide are expressed.
(Item 77)
77. The non-natural cell of any one of items 1-76, wherein said cell is a hematopoietic cell.
(Item 78)
77. The non-natural cell of any one of items 1-76, wherein said cell is a T cell.
(Item 79)
said cells are CD3 ⁺ , CD4 ⁺ and/or CD8 ⁺ 77. The non-natural cell according to any one of items 1-76, which is a cell.
(Item 80)
77. The non-natural cell of any one of items 1-76, wherein said cell is an immune effector cell.
(Item 81)
77. The non-natural cell of any one of items 1-76, wherein said cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.
(Item 82)
77. The non-natural cell of any one of items 1-76, wherein said cell is a natural killer (NK) cell or a natural killer T (NKT) cell.
(Item 83)
83. Any one of items 1 to 82, wherein the source of said cells is peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue or tumor. Non-native cells as described.
(Item 84)
a fusion polypeptide,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a first co-stimulatory domain, and/or a primary signaling domain; and
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising a second co-stimulatory domain .
(Item 85)
85. The fusion polypeptide of item 84, wherein said first and second multimerization domains are different.
(Item 86)
86. The fusion polypeptide of item 84 or item 85, wherein said first and second co-stimulatory domains are different.
(Item 87)
Said first multimerization domain and said second multimerization domain are rapamycin or its rapalogs, coumarmycin or its derivatives, gibberellin or its derivatives, abscisic acid (ABA) or its derivatives, methotrexate or its derivatives, cyclosporine A or a derivative thereof, FK506/cyclosporin A (FKCsA) or a derivative thereof, and a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof. , items 84-86.
(Item 88)
The first multimerization domain and the second multimerization domain are FKBP and FKBP-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin , and a pair selected from the group consisting of PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
(Item 89)
89. The method of any one of items 84-88, wherein the first multimerization domain comprises a FKBP polypeptide or variant thereof and the second multimerization domain comprises a FRB polypeptide or variant thereof. fusion polypeptide.
(Item 90)
89. The method of any one of items 84-88, wherein the first multimerization domain comprises a FRB polypeptide or variant thereof and the second multimerization domain comprises a FKBP polypeptide or variant thereof. fusion polypeptide.
(Item 91)
91. The fusion polypeptide of any one of items 84-90, wherein said cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. .
(Item 92)
92. The fusion polypeptide of any one of items 84-91, wherein said first and second multimerization domains are selected from FRB T2098L and FKBP12 and said cross-linking agent is sirolimus or AP21967.
(Item 93)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, Amnionless (AMN), and programmed cell death 1 (PDCD1) 93. A fusion polypeptide according to any one of items 84-92, selected from selected polypeptides.
(Item 94)
Any of items 84-93, wherein said first transmembrane domain and said second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain. A fusion polypeptide according to one of the paragraphs.
(Item 95)
95. The fusion polypeptide of any one of items 84-94, wherein said first transmembrane domain and said second transmembrane domain are different.
(Item 96)
96. The fusion polypeptide of any one of items 84-95, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 97)
Said first and second co-stimulatory domains are independently Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 ( CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 (DAP10), Ｌｉｎｋｅｒ ｆｏｒ ａｃｔｉｖａｔｉｏｎ ｏｆ Ｔ－ｃｅｌｌｓ ｆａｍｉｌｙ ｍｅｍｂｅｒ １（ＬＡＴ）、ＳＨ２ Ｄｏｍａｉｎ－Ｃｏｎｔａｉｎｉｎｇ Ｌｅｕｋｏｃｙｔｅ Ｐｒｏｔｅｉｎ Ｏｆ ７６ ｋＤ（ＳＬＰ７６）、Ｔ ｃｅｌｌ ｒｅｃｅｐｔｏｒ ａｓｓｏｃｉａｔｅｄ ｔｒａｎｓｍｅｍｂｒａｎｅ ａｄａｐｔｏｒ １（ＴＲＡＴ１）、ＴＮＦＲ２、ＴＮＦＲＳ１４、ＴＮＦＲＳ１８、ＴＮＦＲＳ２５、およびｚｅｔａ ｃｈａｉｎ ｏｆ 97. The fusion polypeptide of any one of items 84-96, selected from costimulatory molecules selected from the group consisting of T cell receptor associated protein kinase 70 (ZAP70).
(Item 98)
said first costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of CD28, CD134 and CD137, and said second costimulatory domain comprises CD28, CD278, TNFRS14, TNFRS18, TNFRS25 , OX40 or TNFR2.
(Item 99)
99. The fusion polypeptide of any one of items 84-98, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from OX40 or TNFR2.
(Item 100)
99. The fusion polypeptide of any one of items 84-99, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from OX40.
(Item 101)
99. The fusion polypeptide of any one of items 84-99, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from TNFR2.
(Item 102)
102. Any one of items 84-101, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. A fusion polypeptide as described in .
(Item 103)
103. The fusion polypeptide of any one of items 84-102, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 104)
104. The fusion polypeptide of any one of items 84-103, wherein said extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.
(Item 105)
The extracellular binding domain may be camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single domain antibodies (sdAbs, camelid VHHs, nanobodies) or antigen-binding fragments thereof. , items 84-104.
(Item 106)
106. The fusion polypeptide of any one of items 84-105, wherein said extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.
(Item 107)
106. The fusion polypeptide of any one of items 84-105, wherein said extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.
(Item 108)
108. The fusion polypeptide of any one of items 84-107, wherein said extracellular binding domain comprises a scFv.
(Item 109)
108. The fusion polypeptide of any one of items 84-107, wherein said extracellular binding domain comprises one or more camelid VHH antibodies.
(Item 110)
wherein said extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand, item A fusion polypeptide according to any one of paragraphs 84-109.
(Item 111)
wherein the extracellular binding domain is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 111. The fusion polypeptide of any one of items 84-110, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 112)
a fusion polypeptide,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain.
(Item 113)
a fusion polypeptide,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising a TNFR2 co-stimulatory domain.
(Item 114)
114. The fusion polypeptide of item 112 or item 113, wherein said first and second multimerization domains are the same.
(Item 115)
114. The fusion polypeptide of item 112 or item 113, wherein said first and second multimerization domains are different.
(Item 116)
Said first multimerization domain and said second multimerization domain are rapamycin or a rapalog thereof, coumarmycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporine A or a derivative thereof , FKCsA or a derivative thereof, and SLF or a derivative thereof.
(Item 117)
said first multimerization domain and said second multimerization domain are selected from the group consisting of FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and DHFR, calcineurin and cyclophilin, and PYL1 and ABI1 A fusion polypeptide according to items 112-116, which is a pair.
(Item 118)
118. The fusion polypeptide of items 112-117, wherein said first multimerization domain comprises a FKBP polypeptide or variant thereof and said second multimerization domain comprises a FRB polypeptide or variant thereof.
(Item 119)
118. The fusion polypeptide of items 112-117, wherein said first multimerization domain comprises a FRB polypeptide or variant thereof and said second multimerization domain comprises a FKBP polypeptide or variant thereof.
(Item 120)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1. 112-119.
(Item 121)
121. The fusion polypeptide of items 112-120, wherein said first transmembrane domain and said second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain and a CD8α transmembrane domain.
(Item 122)
122. The fusion polypeptide of items 112-121, wherein said first transmembrane domain and said second transmembrane domain are identical.
(Item 123)
122. The fusion polypeptide of items 112-121, wherein said first transmembrane domain and said second transmembrane domain are different.
(Item 124)
124. Fusion polypeptide according to items 112-123, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 125)
The co-stimulatory domains are TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, isolated from a co-stimulatory molecule selected from the group consisting of CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70 , items 112-124.
(Item 126)
126. The fusion polypeptide of items 112-125, wherein said co-stimulatory domain is isolated from a co-stimulatory molecule selected from the group consisting of CD28, CD134 and CD137.
(Item 127)
The fusion polypeptide of items 112-126, wherein said co-stimulatory domain is isolated from a CD137 co-stimulatory molecule.
(Item 128)
128. The fusion polypeptide of items 112-127, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. peptide.
(Item 129)
129. The fusion polypeptide of items 112-128, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 130)
The fusion polypeptide of items 112-129, wherein said extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.
(Item 131)
Said extracellular binding domains are camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single domain antibodies (sdAbs, camelid VHHs, nanobodies) or antigen-binding fragments thereof. , items 112-130.
(Item 132)
The fusion polypeptide of items 112-131, wherein said extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.
(Item 133)
The fusion polypeptide of items 112-131, wherein said extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.
(Item 134)
The fusion polypeptide of items 112-133, wherein said extracellular binding domain comprises an scFv.
(Item 135)
132. The fusion polypeptide of items 112-131, wherein said extracellular binding domain comprises one or more camelid VHH antibodies.
(Item 136)
wherein said extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand, item 112-135.
(Item 137)
wherein the extracellular binding domain is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 137. The fusion polypeptide of items 112-136, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 138)
a fusion polypeptide,
(a) a first polypeptide comprising a FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain.
(Item 139)
a fusion polypeptide,
(a) a first polypeptide comprising a FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising a TNFR2 co-stimulatory domain.
(Item 140)
a fusion polypeptide,
(a) a first polypeptide comprising a FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain.
(Item 141)
a fusion polypeptide,
(a) a first polypeptide comprising a FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second polypeptide comprising a TNFR2 co-stimulatory domain.
(Item 142)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1. A fusion polypeptide according to any one of paragraphs 138-141.
(Item 143)
Any of items 138 to 142, wherein said first transmembrane domain and said second transmembrane domain are independently selected from polypeptides selected from the group consisting of a CD4 transmembrane domain and a CD8α transmembrane domain. A fusion polypeptide according to one of the paragraphs.
(Item 144)
144. The fusion polypeptide of any one of items 138-143, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 145)
The co-stimulatory domains are TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, isolated from a co-stimulatory molecule selected from the group consisting of CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2, TNFRS14, TNFRS18, TNFRS25, and ZAP70 , items 138-144.
(Item 146)
146. The fusion polypeptide of any one of items 138-145, wherein said co-stimulatory domain is isolated from a co-stimulatory molecule selected from the group consisting of CD28, CD134 and CD137.
(Item 147)
147. The fusion polypeptide of any one of items 138-146, wherein said costimulatory domain is isolated from the CD137 costimulatory molecule.
(Item 148)
148. Any one of items 138-147, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. A fusion polypeptide as described in .
(Item 149)
149. The fusion polypeptide of any one of items 138-148, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 150)
The antibody or antigen-binding fragment thereof is camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (“scFv”), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (“dsFv”), and single domain antibodies (sdAb, camelid VHH, nanobodies) of items 138-149. A fusion polypeptide according to any one of paragraphs.
(Item 151)
151. The fusion polypeptide of any one of items 138-150, wherein said antibody or antigen-binding fragment thereof is human or humanized.
(Item 152)
151. The fusion polypeptide of any one of items 138-150, wherein said antibody or antigen-binding fragment thereof comprises a scFv or one or more camelid VHH antibodies.
(Item 153)
The antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand. , items 138-152.
(Item 154)
The antibody or antigen-binding fragment thereof is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 154. The fusion polypeptide of any one of items 138-153, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 155)
155. The fusion polypeptide of any one of items 138-154, wherein said antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B and/or EGFRvIII.
(Item 156)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to BCMA.
(Item 157)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to CD19.
(Item 158)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to CD20 or CD22.
(Item 159)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to B7-H3.
(Item 160)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to CD33.
(Item 161)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to CD79A.
(Item 162)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to CD79B.
(Item 163)
156. The fusion polypeptide of any one of items 138-155, wherein said antibody or antigen-binding fragment thereof binds to EGFRvIII.
(Item 164)
a fusion polypeptide,
(a) a first polypeptide comprising a FKBP12 multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB T2098L multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a second polypeptide comprising a TNFR2 co-stimulatory domain.
(Item 165)
a fusion polypeptide,
(a) a first polypeptide comprising a FKBP12 multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB T2098L multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain.
(Item 166)
a fusion polypeptide,
(a) a first polypeptide comprising a FRB T2098L multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP12 multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a second polypeptide comprising a TNFR2 co-stimulatory domain.
(Item 167)
a fusion polypeptide,
(a) a first polypeptide comprising a FRB T2098L multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) a polypeptide cleavage signal; and
(c) a fusion polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP12 multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a second polypeptide comprising an OX40 co-stimulatory domain.
(Item 168)
The antibody or antigen-binding fragment thereof is camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (“scFv”), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (“dsFv”), and single domain antibodies (sdAb, camelid VHH, nanobodies) of items 164-167. A fusion polypeptide according to any one of paragraphs.
(Item 169)
169. The fusion polypeptide of any one of items 164-168, wherein said antibody or antigen-binding fragment thereof is human or humanized.
(Item 170)
169. The fusion polypeptide of any one of items 164-169, wherein said antibody or antigen-binding fragment thereof comprises a scFv or one or more camelid VHH antibodies.
(Item 171)
The antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand. , items 164-170.
(Item 172)
The antibody or antigen-binding fragment thereof is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 172. The fusion polypeptide of any one of items 164-171, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 173)
173. The fusion polypeptide of any one of items 164-172, wherein said antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B and/or EGFRvIII.
(Item 174)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds to BCMA.
(Item 175)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds to CD19.
(Item 176)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds to CD20 or CD22.
(Item 177)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds to B7-H3.
(Item 178)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds to CD33.
(Item 179)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds to CD79A.
(Item 180)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds to CD79B.
(Item 181)
174. The fusion polypeptide of any one of items 164-173, wherein said antibody or antigen-binding fragment thereof binds EGFRvIII, optionally said antibody is EGFR806 or an antigen-binding fragment thereof.
(Item 182)
182. The fusion polypeptide of any one of items 164-181, wherein said multimerization domain is located extracellularly when said first polypeptide and said second polypeptide are expressed.
(Item 183)
183. The fusion polypeptide of any one of items 84-182, wherein said polypeptide cleavage signal is a viral self-cleaving polypeptide.
(Item 184)
184. The fusion polypeptide of any one of items 84-183, wherein said polypeptide cleavage signal is a viral self-cleaving 2A polypeptide.
(Item 185)
The polypeptide cleavage signals include foot and mouth disease virus (FMDV) (F2A) peptide, equine rhinitis A virus (ERAV) (E2A) peptide, Thosea asigna virus (TaV) (T2A) peptide, porcine tessho virus-1 (PTV-1 185. The fusion polypeptide of any one of items 84-184, which is a viral self-cleaving polypeptide selected from the group consisting of ) (P2A) peptide, tyrovirus 2A peptide, and encephalomyocarditis virus 2A peptide.
(Item 186)
A polynucleotide encoding the first or second polypeptide according to any one of items 1-83 or encoding the fusion polypeptide according to any one of items 84-185.
(Item 187)
A cDNA encoding the first or second polypeptide according to any one of items 1-83 or encoding the fusion polypeptide according to any one of items 84-185.
(Item 188)
RNA encoding the first or second polypeptide according to any one of items 1-83 or encoding the fusion polypeptide according to any one of items 84-185.
(Item 189)
A vector containing a polynucleotide according to any one of items 186-188.
(Item 190)
189. The vector of item 189, wherein said vector is an expression vector.
(Item 191)
189. The vector of item 189, wherein said vector is a transposon.
(Item 192)
192. The vector of item 191, wherein said vector is a piggyBAC transposon or a Sleeping Beauty transposon.
(Item 193)
189. The vector of item 189, wherein said vector is a viral vector.
(Item 194)
194. The vector of item 193, wherein said vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a vaccinia viral vector, or a retroviral vector.
(Item 195)
195. The vector of item 194, wherein said retroviral vector is a lentiviral vector.
(Item 196)
The lentiviral vector is human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV) virus, goat arthritis encephalitis virus (CAEV), equine infectious anemia 196. The vector of item 195, selected from the group consisting of viruses (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).
(Item 197)
the non-natural cell of any one of items 1-83, the fusion polypeptide of any one of items 84-185, the polynucleotide of any one of items 186-188, or item 189 A composition comprising a vector according to any one of paragraphs 1-96.
(Item 198)
a pharmaceutically acceptable carrier and the non-natural cell of any one of items 1-83, the fusion polypeptide of any one of items 84-185, any one of items 186-188 or a vector according to any one of items 189-196.
(Item 199)
A method of treating a subject in need thereof, comprising administering to said subject an effective amount of the composition of item 197 or item 198.
(Item 200)
198. A method of treating, preventing or ameliorating at least one symptom of, or conditions associated with, cancer, infectious diseases, autoimmune diseases, inflammatory diseases and immunodeficiencies, the composition according to item 197 or item 198 administering to said subject an effective amount of a substance.
(Item 201)
A method of treating a solid tumor, comprising administering to said subject an effective amount of the composition of item 197 or item 198.
(Item 202)
The solid cancer includes liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer. 201.
(Item 203)
203. The method of item 201 or 202, wherein said solid cancer is pancreatic cancer, lung cancer or breast cancer.
(Item 204)
A method of treating a hematological malignancy comprising administering an effective amount of the composition of item 197 or item 198 to said subject.
(Item 205)
205. The method of item 204, wherein said hematologic malignancy is leukemia, lymphoma or multiple myeloma.
(Item 206)
A polypeptide complex,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a first co-stimulatory domain, and/or a primary signaling domain; and
(b) a second polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 207)
207. The polypeptide complex of item 206, wherein said first and second multimerization domains are different.
(Item 208)
208. The polypeptide complex of item 206 or item 207, wherein said first and second co-stimulatory domains are different.
(Item 209)
Said first multimerization domain and said second multimerization domain are rapamycin or its rapalogs, coumarmycin or its derivatives, gibberellin or its derivatives, abscisic acid (ABA) or its derivatives, methotrexate or its derivatives, cyclosporine A or a derivative thereof, FK506/cyclosporin A (FKCsA) or a derivative thereof, and a synthetic ligand (SLF) of trimethoprim (Tmp)-FK506 binding protein (FKBP) or a derivative thereof. , items 206-208.
(Item 210)
The first multimerization domain and the second multimerization domain are FKBP and FKBP-rapamycin binding (FRB), FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial dihydrofolate reductase (DHFR), calcineurin and cyclophilin , and a pair selected from the group consisting of PYR1-like 1 (PYL1) and abscisic acid insensitive 1 (ABI1).
(Item 211)
211. The method of any one of items 206-210, wherein said first multimerization domain comprises a FKBP polypeptide or variant thereof and said second multimerization domain comprises a FRB polypeptide or variant thereof. Polypeptide complex.
(Item 212)
The method of any one of items 206-211, wherein said first multimerization domain comprises a FRB polypeptide or variant thereof and said second multimerization domain comprises a FKBP polypeptide or variant thereof. Polypeptide complex.
(Item 213)
213. The polypeptide conjugate of any one of items 206-212, wherein said cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. body.
(Item 214)
214. The polypeptide complex of any one of items 206-213, wherein said first and second multimerization domains are selected from FRB T2098L and FKBP12 and said cross-linking agent is sirolimus or AP21967.
(Item 215)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, Amnionless (AMN), and programmed cell death 1 (PDCD1) 215. A polypeptide complex according to any one of items 206-214, selected from selected polypeptides.
(Item 216)
Any of items 206-215, wherein said first transmembrane domain and said second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain A polypeptide conjugate according to any one of the preceding paragraphs.
(Item 217)
217. The polypeptide complex of any one of items 206-216, wherein said first transmembrane domain and said second transmembrane domain are different.
(Item 218)
218. Polypeptide complex according to any one of items 206-217, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 219)
The first and second co-stimulatory domains are independently Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family
member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation
Protein 10 (DAP10), Linker for activation
ｏｆ Ｔ－ｃｅｌｌｓ ｆａｍｉｌｙ ｍｅｍｂｅｒ １（ＬＡＴ）、ＳＨ２ Ｄｏｍａｉｎ－Ｃｏｎｔａｉｎｉｎｇ Ｌｅｕｋｏｃｙｔｅ Ｐｒｏｔｅｉｎ Ｏｆ ７６ ｋＤ（ＳＬＰ７６）、Ｔ ｃｅｌｌ ｒｅｃｅｐｔｏｒ ａｓｓｏｃｉａｔｅｄ ｔｒａｎｓｍｅｍｂｒａｎｅ ａｄａｐｔｏｒ １（ＴＲＡＴ１）、ＴＮＦＲ２、ＴＮＦＲＳ１４、ＴＮＦＲＳ１８、ＴＮＦＲＳ２５、およびｚｅｔａ ｃｈａｉｎ ｏｆ Ｔ ｃｅｌｌ
219. The polypeptide complex of any one of items 206-218, isolated from a costimulatory molecule selected from the group consisting of receptor associated protein kinase 70 (ZAP70).
(Item 220)
said first costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of CD28, CD134 and CD137, and said second costimulatory domain comprises CD28, CD278, TNFRS14, TNFRS18, TNFRS25 219. The polypeptide complex of any one of items 206-219, which is isolated from , OX40 or TNFR2.
(Item 221)
221. The polypeptide complex of any one of items 206-220, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from OX40 or TNFR2 .
(Item 222)
222. The polypeptide complex of any one of items 206-221, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from OX40.
(Item 223)
222. The polypeptide complex of any one of items 206-221, wherein said first costimulatory domain is isolated from CD137 and said second costimulatory domain is isolated from TNFR2.
(Item 224)
Any one of items 206-223, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. The polypeptide conjugate according to .
(Item 225)
225. The polypeptide complex of any one of items 206-224, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 226)
226. The polypeptide complex of any one of items 206-225, wherein said extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.
(Item 227)
The extracellular binding domain may be camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single domain antibodies (sdAbs, camelid VHHs, nanobodies) or antigen-binding fragments thereof. , items 206-226.
(Item 228)
228. The polypeptide complex of any one of items 206-227, wherein said extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.
(Item 229)
228. The polypeptide complex of any one of items 206-227, wherein said extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.
(Item 230)
230. The polypeptide complex of any one of items 206-229, wherein said extracellular binding domain comprises an scFv.
(Item 231)
230. The polypeptide complex of any one of items 206-229, wherein said extracellular binding domain comprises one or more camelid VHH antibodies.
(Item 232)
wherein said extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand, item 231. The polypeptide complex according to any one of 206-231.
(Item 233)
wherein the extracellular binding domain is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 233. The polypeptide complex according to any one of items 206-232, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 234)
A polypeptide complex,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a second polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and an OX40 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 235)
A polypeptide complex,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a second polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a TNFR2 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 236)
236. The polypeptide complex of item 234 or item 235, wherein said first and second multimerization domains are the same.
(Item 237)
237. The polypeptide complex of item 234 or item 236, wherein said first and second multimerization domains are different.
(Item 238)
Said first multimerization domain and said second multimerization domain are rapamycin or a rapalog thereof, coumarmycin or a derivative thereof, gibberellin or a derivative thereof, ABA or a derivative thereof, methotrexate or a derivative thereof, cyclosporine A or a derivative thereof , FKCsA or a derivative thereof, and SLF or a derivative thereof, associated with a cross-linking agent.
(Item 239)
said first multimerization domain and said second multimerization domain are selected from the group consisting of FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and DHFR, calcineurin and cyclophilin, and PYL1 and ABI1 239. The polypeptide complex according to any one of items 234-238, which is a pair.
(Item 240)
The method of any one of items 234-239, wherein said first multimerization domain comprises a FKBP polypeptide or variant thereof and said second multimerization domain comprises a FRB polypeptide or variant thereof. Polypeptide complex.
(Item 241)
The method of any one of items 234-239, wherein said first multimerization domain comprises a FRB polypeptide or variant thereof and said second multimerization domain comprises a FKBP polypeptide or variant thereof. Polypeptide complex.
(Item 242)
242. The polypeptide conjugate of any one of items 234-241, wherein said cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. body.
(Item 243)
243. The polypeptide complex of any one of items 234-242, wherein said first and second multimerization domains are selected from FRB T2098L and FKBP12 and said cross-linking agent is sirolimus or AP21967.
(Item 244)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1. The polypeptide complex according to any one of 234-243.
(Item 245)
245. The poly of any one of items 234-244, wherein said first transmembrane domain and said second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain and a CD8α transmembrane domain. peptide complex.
(Item 246)
246. The polypeptide complex of any one of items 234-245, wherein said first transmembrane domain and said second transmembrane domain are the same.
(Item 247)
246. The polypeptide complex of any one of items 234-245, wherein said first transmembrane domain and said second transmembrane domain are different.
(Item 248)
248. Polypeptide complex according to any one of items 234-247, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 249)
The co-stimulatory domains are TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, Any of items 234-248 isolated from a co-stimulatory molecule selected from the group consisting of CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2 and ZAP70 or the polypeptide complex according to claim 1.
(Item 250)
250. The polypeptide complex of any one of items 234-249, wherein said costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of CD28, CD134 and CD137.
(Item 251)
251. The polypeptide conjugate of any one of items 234-250, wherein said costimulatory domain is isolated from a CD137 costimulatory molecule.
(Item 252)
Any one of items 234-251, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. The polypeptide conjugate according to .
(Item 253)
253. The polypeptide complex of any one of items 234-252, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 254)
254. The polypeptide complex of any one of items 234-253, wherein said extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand.
(Item 255)
The extracellular binding domain may be camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single domain antibodies (sdAbs, camelid VHHs, nanobodies) or antigen-binding fragments thereof. , items 234-254.
(Item 256)
256. The polypeptide complex of any one of items 234-255, wherein said extracellular binding domain comprises a humanized antibody or antigen-binding fragment thereof.
(Item 257)
256. The polypeptide complex of any one of items 234-255, wherein said extracellular binding domain comprises a human antibody or antigen-binding fragment thereof.
(Item 258)
258. The polypeptide complex of any one of items 234-257, wherein said extracellular binding domain comprises a scFv.
(Item 259)
256. The polypeptide complex of any one of items 234-255, wherein said extracellular binding domain comprises one or more camelid VHH antibodies.
(Item 260)
wherein said extracellular binding domain binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand, item 259. The polypeptide complex according to any one of 234-259.
(Item 261)
wherein the extracellular binding domain is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 261. The polypeptide complex of any one of items 234-260, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 262)
A polypeptide complex,
(a) a first polypeptide comprising a FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB multimerization domain polypeptide or variant thereof, a second transmembrane domain, and an OX40 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 263)
A polypeptide complex,
(a) a first polypeptide comprising a FKBP multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a TNFR2 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 264)
A polypeptide complex,
(a) a first polypeptide comprising a FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP multimerization domain polypeptide or variant thereof, a second transmembrane domain, and an OX40 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 265)
A polypeptide complex,
(a) a first polypeptide comprising a FRB multimerization domain polypeptide or variant thereof, a first transmembrane domain, a co-stimulatory domain, and/or a primary signaling domain;
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a TNFR2 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 266)
266. The polypeptide conjugate of any one of items 262-265, wherein said cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. body.
(Item 267)
267. The polypeptide complex of any one of items 262-266, wherein the FRB multimerization domain is FRB T2098L, the FKBP multimerization domain is FKBP12, and the cross-linking agent is sirolimus or AP21967.
(Item 268)
Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN, and PDCD1. The polypeptide complex according to any one of 262-267.
(Item 269)
Any of items 262-268, wherein said first transmembrane domain and said second transmembrane domain are independently selected from a polypeptide selected from the group consisting of a CD4 transmembrane domain and a CD8α transmembrane domain A polypeptide conjugate according to any one of the preceding paragraphs.
(Item 270)
269. Polypeptide complex according to any one of items 262-269, wherein said co-stimulatory domain and/or said primary signaling domain contains an immunoreceptor tyrosine activation motif (ITAM).
(Item 271)
The co-stimulatory domains are TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, Any of items 262-270 isolated from a co-stimulatory molecule selected from the group consisting of CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, SLP76, TRAT1, TNFR2 and ZAP70 or the polypeptide complex according to claim 1.
(Item 272)
272. The polypeptide complex of any one of items 262-271, wherein said costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of CD28, CD134 and CD137.
(Item 273)
273. The polypeptide conjugate according to any one of items 262-272, wherein said costimulatory domain is isolated from a CD137 costimulatory molecule.
(Item 274)
Any one of items 262-273, wherein said primary signaling domain is isolated from a polypeptide selected from the group consisting of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. The polypeptide conjugate according to .
(Item 275)
275. The polypeptide complex of any one of items 262-274, wherein said primary signaling domain is isolated from a CD3ζ polypeptide.
(Item 276)
The antibody or antigen-binding fragment thereof is camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (“scFv”), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (“dsFv”), and single domain antibodies (sdAb, camelid VHH, nanobodies) of items 262-275. A polypeptide conjugate according to any one of claims.
(Item 277)
277. The polypeptide conjugate of any one of items 262-276, wherein said antibody or antigen-binding fragment thereof is human or humanized.
(Item 278)
278. The polypeptide complex of any one of items 262-277, wherein said antibody or antigen-binding fragment thereof comprises a scFv or one or more camelid VHH antibodies.
(Item 279)
The antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand. , items 262-278.
(Item 280)
The antibody or antigen-binding fragment thereof is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 279. The polypeptide complex according to any one of items 262-279, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 281)
281. The polypeptide complex of any one of items 262-280, wherein said antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B and/or EGFRvIII. .
(Item 282)
282. The polypeptide conjugate of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to BCMA.
(Item 283)
282. The polypeptide conjugate of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to CD19.
(Item 284)
282. The polypeptide complex of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to CD20 or CD22.
(Item 285)
282. The polypeptide complex of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to B7-H3.
(Item 286)
282. The polypeptide conjugate of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to CD33.
(Item 287)
282. The polypeptide complex of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to CD79A.
(Item 288)
282. The polypeptide conjugate of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to CD79B.
(Item 289)
282. The polypeptide complex of any one of items 262-281, wherein said antibody or antigen-binding fragment thereof binds to EGFRvIII.
(Item 290)
A polypeptide complex,
(a) a first polypeptide comprising a FKBP12 multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FRB T2098L multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a TNFR2 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 291)
A polypeptide complex,
(a) a first polypeptide comprising a FKBP12 multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) an antibody or antigen-binding fragment thereof, a FRB T2098L multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and an OX40 co-stimulatory domain;
a second polypeptide comprising, and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 292)
A polypeptide complex,
(a) a first polypeptide comprising a FRB T2098L multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP12 multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and a TNFR2 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 293)
A polypeptide complex,
(a) a first polypeptide comprising a FRB T2098L multimerization domain polypeptide or variant thereof, a CD8α transmembrane domain, a CD137 co-stimulatory domain, and a CD3ζ primary signaling domain;
(b) a second polypeptide comprising an antibody or antigen-binding fragment thereof, a FKBP12 multimerization domain polypeptide or variant thereof, a CD4 transmembrane domain, and an OX40 co-stimulatory domain; and
(c) a polypeptide complex comprising a cross-linking factor associated with and disposed between the multimerization domains of said first and second polypeptides.
(Item 294)
294. The polypeptide complex of any one of items 290-293, wherein said cross-linking agent is AP21967 or sirolimus.
(Item 295)
The antibody or antigen-binding fragment thereof is camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv protein (“scFv”), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (“dsFv”), and single domain antibodies (sdAb, camelid VHH, nanobodies) of items 290-294. A polypeptide conjugate according to any one of claims.
(Item 296)
296. The polypeptide conjugate of any one of items 290-295, wherein said antibody or antigen-binding fragment thereof is human or humanized.
(Item 297)
297. The polypeptide complex of any one of items 290-296, wherein said antibody or antigen-binding fragment thereof comprises a scFv or one or more camelid VHH antibodies.
(Item 298)
The antibody or antigen-binding fragment thereof binds to an antigen selected from the group consisting of a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), an NKG2D ligand, a γδ T cell receptor (TCR) ligand, and an αβTCR ligand. , items 290-297.
(Item 299)
The antibody or antigen-binding fragment thereof is FRα, α _V β ₆ Integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CEA, CLDN6, CLDN18.2, CLL-1, CS-1, CSPG4, CTAGE1, DLL3, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, EPHA2, ERBB4, FAP, FCRL5, AchR, GD2, GD3, GPC3, HER2, HER2 p95, IL-10Rα, IL-13Rα2, Kappa, LAGE-1A, Lambda, LeY, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, MelanA or MART1, SLN ), MUC1, MUC16, MICA, MICB, NCAM, NY-ESO-1, PLAC1, PRAME, PSCA, PSMA, ROR1, SSX2, Survivin, TAG72, TEM1/CD248, TEM7R, TPBG, ULBP1, ULBP2, ULBP3, ULBP4, 299. The polypeptide complex according to any one of items 290-298, which binds to an antigen selected from the group consisting of ULBP5, ULBP6, VEGFR2 and WT-1.
(Item 300)
299. The polypeptide complex of any one of items 290-299, wherein said antibody or antigen-binding fragment thereof binds to BCMA, B7-H3, CD19, CD20, CD22, CD33, CD79A, CD79B and/or EGFRvIII. .
(Item 301)
301. The polypeptide conjugate of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds to BCMA.
(Item 302)
301. The polypeptide complex of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds to CD19.
(Item 303)
301. The polypeptide complex of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds to CD20 or CD22.
(Item 304)
301. The polypeptide complex of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds to B7-H3.
(Item 305)
301. The polypeptide complex of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds to CD33.
(Item 306)
301. The polypeptide complex of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds to CD79A.
(Item 307)
301. The polypeptide conjugate of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds to CD79B.
(Item 308)
301. The polypeptide complex of any one of items 290-300, wherein said antibody or antigen-binding fragment thereof binds EGFRvIII, optionally said antibody is EGFR806 or an antigen-binding fragment thereof.
(Item 309)
301. The polypeptide complex according to any one of items 290-300, wherein said multimerization domain is located extracellularly when said first polypeptide and said second polypeptide are expressed.

Claims (21)

a non-natural cell,
(a) a first polypeptide comprising a first multimerization domain polypeptide or variant thereof, a first transmembrane domain, a first co-stimulatory domain, and/or a primary signaling domain; and (b) a second polypeptide comprising an extracellular binding domain, a second multimerization domain polypeptide or variant thereof, a second transmembrane domain, and a second co-stimulatory domain;
A bridging factor promotes formation of a polypeptide complex on said non-native cell surface, said bridging factor being associated with and disposed between said first and second multimerization domains. natural cells. 2. The non-natural cell of claim 1, wherein said first and second multimerization domains are different. 3. The non-natural cell of claim 1 or claim 2, wherein said first and second co-stimulatory domains are different. (i) said first multimerization domain comprises a FKBP polypeptide or variant thereof and said second multimerization domain comprises a FRB polypeptide or variant thereof, or
(ii) said first multimerization domain comprises a FRB polypeptide or variant thereof and said second multimerization domain comprises a FKBP polypeptide or variant thereof;
The non-natural cell according to any one of claims 1-3 . 5. The non-natural according to any one of claims 1 to 4 , wherein the cross-linking agent is selected from the group consisting of AP21967, sirolimus, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus. cell. 6. The non-natural cell of any one of claims 1-5 , wherein said first and second multimerization domains are selected from FRB T2098L and FKBP12 and said cross-linking agent is sirolimus or AP21967. Said first transmembrane domain and said second transmembrane domain are independently a T cell receptor alpha, beta, gamma or delta chain, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD4, CD5, CD8alpha. , CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, CD278, amnionless (AMN), and programmed cell death 1 (PDCD1) Non-natural cell according to any one of claims 1 to 6 , selected from polypeptides selected from the group. The first transmembrane domain and the second transmembrane domain are independently selected from the group consisting of a CD4 transmembrane domain, a CD8α transmembrane domain, and an AMN transmembrane domain. or the non-natural cell according to item 1. The non-natural cell of any one of claims 1-8 , wherein said first transmembrane domain and said second transmembrane domain are different. (i) said first and second co-stimulatory domains are independently Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-Activation Protein 10 ( ＤＡＰ１０）、Ｌｉｎｋｅｒ ｆｏｒ ａｃｔｉｖａｔｉｏｎ ｏｆ Ｔ－ｃｅｌｌｓ ｆａｍｉｌｙ ｍｅｍｂｅｒ １（ＬＡＴ）、ＳＨ２ Ｄｏｍａｉｎ－Ｃｏｎｔａｉｎｉｎｇ Ｌｅｕｋｏｃｙｔｅ Ｐｒｏｔｅｉｎ Ｏｆ ７６ ｋＤ（ＳＬＰ７６）、Ｔ ｃｅｌｌ ｒｅｃｅｐｔｏｒ ａｓｓｏｃｉａｔｅｄ ｔｒａｎｓｍｅｍｂｒａｎｅ ａｄａｐｔｏｒ １（ＴＲＡＴ１）、ＴＮＦＲ２、ＴＮＦＲＳ１４、ＴＮＦＲＳ１８、ＴＮＦＲＳ２５、およびis selected from a co-stimulatory molecule selected from the group consisting of zeta chain of T cell receptor associated protein kinase 70 (ZAP70), or
(ii) said first costimulatory domain is isolated from a costimulatory molecule selected from the group consisting of CD28, CD134 and CD137, and said second costimulatory domain comprises CD28, CD278, TNFRS14, isolated from TNFRS18, TNFRS25, OX40 or TNFR2;
The non-natural cell according to any one of claims 1-9 . 11. The non-natural cell of any one of claims 1-10 , wherein the first costimulatory domain is isolated from CD137 and the second costimulatory domain is isolated from OX40 or TNFR2. . 12. The non-natural cell of any one of claims 1-11 , wherein said primary signaling domain is isolated from a CD3ζ polypeptide. 13. The non-natural cell of any one of claims 1-12 , wherein said extracellular binding domain comprises an antibody or antigen-binding fragment thereof, a receptor extracellular domain, or a ligand. Said extracellular binding domains are camelid Ig, llama Ig, alpaca Ig, Ig NAR, Fab' fragment, F(ab') ₂ fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer ( Fab3), Fv, single-chain Fv proteins (scFv), bis-scFv, (scFv) ₂ , minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), and single domain antibodies (sdAb , Camelidae VHH, Nanobodies ) or antigen-binding fragment thereof. 15. The non-natural cell of any one of claims 1-14 , wherein said extracellular binding domain comprises one or more camelid VHH antibodies. The extracellular binding domains are alpha folate receptor (FRα), α _v β ₆ integrin, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H6, carbonic anhydrase type IX (CAIX), CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, carcinoembryonic antigen (CEA), claudin-6 ( CLDN6), claudin 18 isoform 2 (CLDN18.2), C-type lectin-like molecule-1 (CLL-1), CD2 subset 1 (CS-1), chondroitin sulfate proteoglycan 4 (CSPG4), cutaneous T cell lymphoma-associated antigen 1 (CTAGE1), delta like canonical Notch ligand 3 (DLL3), epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EPHA2), erb-b2 receptor tyrosine kinase 4 (ERBB4), fibroblast activation protein (FAP), Fc Receptor Like 5, FCRLike 5) EGFR family including acetylcholinesterase receptor (AchR), ganglioside G2 (GD2), ganglioside G3 (GD3), glypican-3 (GPC3), ErbB2 (HER2), HER2 p95, IL-10Rα, IL-13Rα2, Kappa, cancer/testis antigen 2 (LAGE-1A), Lambda, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), melanoma antigen gene (MAGE)-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGEA10, m elanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MUC1, MUC16, MHC class I chain related proteins A (MICA), MHC class I chain related protein B (MICB MCA) ), cancer/testis antigen 1 (NY-ESO-1), polysialic acid; placenta-specific 1 (PLAC1), preferentially expressed antigen in melanoma (PRAME), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA ）、ｒｅｃｅｐｔｏｒ ｔｙｒｏｓｉｎｅ ｋｉｎａｓｅ－ｌｉｋｅ ｏｒｐｈａｎ ｒｅｃｅｐｔｏｒ １（ＲＯＲ１）、ｓｙｎｏｖｉａｌ ｓａｒｃｏｍａ、Ｘ ｂｒｅａｋｐｏｉｎｔ ２（ＳＳＸ２）、サバイビン、ｔｕｍｏｒ ａｓｓｏｃｉａｔｅｄ ｇｌｙｃｏｐｒｏｔｅｉｎ ７２（ＴＡＧ７２）、ｔｕｍｏｒ ｅｎｄｏｔｈｅｌｉａｌ ｍａｒｋｅｒ １（ＴＥＭ１／ＣＤ２４８）、ｔｕｍｏｒ ｅｎｄｏｔｈｅｌｉａｌ ｍａｒｋｅｒ ７－ｒｅｌａｔｅｄ (TEM7R), trophoblast glycoprotein (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, vascular endothelial cell growth factor receptor 2 (VEGFR2), and Wilms tumor 1 (WT-1) 16. The non-natural cell of any one of claims 1-15 , which binds to an antigen selected from the group consisting of: A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the non-natural cell according to any one of claims 1-16 . 18. A pharmaceutical composition according to claim 17 for use in treating , preventing or ameliorating at least one symptom of cancer or an autoimmune disease or a condition associated therewith. wherein said cancer comprises liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, or skin cancer. Item 19. The pharmaceutical composition according to Item 18 . 18. A pharmaceutical composition according to claim 17 for use in the treatment of hematologic malignancies. 21. The pharmaceutical composition according to claim 20 , wherein said hematologic malignancy is leukemia, lymphoma or multiple myeloma.